The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines As Metal Ligands

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Chemistry Dissertations Department of Chemistry

Fall 11-19-2013

The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines as Metal Ligands

Yan Chen

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THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT

UTILIZE THREE HISTIDINES AS METAL LIGANDS

Yan Chen

Under the Direction of Dr. Aimin Liu

ABSTRACT

The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electronic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with

3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure.

The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the disserta-

tion is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase

(ACMSD). ACMSD catalyzes the decarboxylation through a hydrolase-like mechanism that is initialized by the deprotonation of metal bounded water molecule. Our study reveled that the second sphere residue His228 is responsible for the water deprotonation through hydrogen bonding. The spectroscopic and crystallographic data showed the H228Y mutation binds ferric iron instead of native zinc metal and the active site water is replaced by the Tyr228 residue ligation.

Thus, we concluded that, H228Y not only plays a role of stabilizing and deprotonating the active site water but also is an essential residue on metal selectivity.

INDEX WORDS: Cysteamine dioxygenase, Cysteine dioxygenase, ACMSD, Metal binding motif, Metal selectivity, Substrate specificity

THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT

UNLILZE THREE HISTIDINES AS METAL LIGANDS

Yan Chen

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of philosophy

in the College of Arts and Sciences

Georgia State University

2013

Yan Chen 2013

THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT

UNLILZE THREE HISTIDINES AS METAL LIGANDS

Yan Chen

Committee Chair: Dr. Aimin Liu

Committee: Dr. Jenny J. Yang

Dr. Binghe Wang

Electronic Version Approved:

Office of Graduate Studies

College of Arts and Sciences

Georgia State University

December 2013

DEDICATION

I dedicate my dissertation to my deeply loved parents Chen, Weimin and Wang, Yaofeng.

ACKNOWLEDGEMENTS

The result obtained in the dissertation is a team work of a lot of great scientists. I appreciate my supervisor Dr. Aimin Liu for giving me the opportunity to accomplish my research in his laboratory. I was majored in biology technology in my undergraduate school then switched to plan physiology in the graduate program in Lanzhou University. I wasn’t aware of protein purification, EPR or protein crystallization when I came to GSU in 2008. It was Dr. Liu who had spent every Saturday morning in 2008 to teach me the knowledge and the skills that needed for my

Ph.D. research. I appreciate his support and training very much and I will never forget the protein purification, EPR skills and the experimental tricks that he has taught me hand by hand.

I would like to thank Dr. Jenny J.Yang who is always passionate and positive when she talks to me. I will always be encouraged by her positive attitude. I would like to thank Dr. Da- vidson who is a great scientist with strong scientific logics. I have learned a lot from the collabo- ration with him on MauG project. I also want to give my big thanks to Dr. Binghe Wang. Dr.

Wang helped me scientifically as being my committee member. Dr. Wang also gave me unfor- gettable suggestions in my Ph.D. career. I have encountered many people in the past 5 years.

Never will I forget Dr. Rong Fu, Dr. Sunil Naik, Dr. Lirong Chen and all my colleagues for their generous help during my Ph.D. study.

In the past five years, I have learned the most that I can for my future. No matter how time flies, the good things and memories in Dr. Aimin Liu’s laboratory will never fade.

TABLE OF CONTENTS

THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES

THAT UTILIZE THREE HISTIDINES AS METAL LIGANDS ...... i

THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES

THAT UNLILZE THREE HISTIDINES AS METAL LIGANDS ...... iii

THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES

THAT UNLILZE THREE HISTIDINES AS METAL LIGANDS ...... v

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

1 INTRODUCTION...... 1

1.1 Enzymes with one histidine metal ligand ...... 2

1.2 Enzymes with two histidine metal ligands ...... 3

1.2.1 Ring cleavage dioxygenases—Extradiol and Intradiol dioxygenases ...... 4

1.2.2 Fe/ α- ketoglutarate( αKG) dependent enzymes ...... 9

1.2.3 Antibiotic biosynthesis related enzyme- Isopenicillin N synthase ...... 12

1.3 Enzymes with three histidine metal ligands ...... 14

1.3.1 Dioxygenases with 3-His-1-carboxylate motif in cupin superfamily ...... 14

1.3.2 Dioxygenase with unique 3-His iron binding motif in cupin superfamily ...... 18

1.3.3 3-His-1-carboxylate containing enzymes amidohydrolase superfamily ...... 22

1.3.4 Α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) ...... 26

vii

1.3.5 Superoxide dismutase with 3-His-1carboxylate motif ...... 28

1.4 Enzymes with four histidine metal ligands ...... 30

1.4.1 Cu/Zn binuclear SOD ...... 30

1.4.2 Superoxide reductase ...... 31

1.5 The overview of the dissertation ...... 34

2 MATERIAL AND METHODS ...... 36

2.1 ADO and CDO ...... 36

2.1.1 The over-expression and purification of ADO...... 36

2.1.2 Mössbauer spectroscopy study of ADO...... 37

2.1.3 EPR spectroscopy of ADO ...... 38

2.1.4 ADO ESI mass spectrometer sample preparation ...... 38

2.1.5 ADO crystallization ...... 39

2.1.6 Inhibition of the cysteine and homocysteine to ADO ...... 40

2.1.7 DTNB treatment on ADO ...... 41

2.2 Crystallization condition of the Fe, Co-ACMSD ...... 41

3 SPECTROSPCOPIC, STRUCTURAL ANS MECHANISMTIC

CHARATERIZATION OF THE THIOL DIOXYGENASES ...... 43

3.1 EPR and Mössbauer characterization of cysteamine dioxygenase ...... 43

3.1.1 Introduction ...... 44

3.1.2 Materials and methods ...... 46

viii

3.1.3 Result 49

3.1.4 Discussion 57

3.2 The structure characterization of the ADO...... 60

3.2.1 Crystallization of a de novo crystal structure of a mononuclear Non-heme Iron ...... 60

3.2.2 Characterization of the role of cysteine in ADO ...... 61

4 THE INVESTIGATION OF METAL AND SUBSTRATE SPECIFICITY OF

THIOL DIOXYGENASE ...... 67

4.1 ADO is not a iron dependent non-heme enzyme ...... 67

4.1.1.1 Introduction ...... 67

4.2 Materials and Methods ...... 70

4.2.2 Results 71

4.2.3 Discussion 77

4.3 Initial characterization of human enzymes ...... 78

5 THE STRUCTURE AND FUNCTION STUDY OF THE SPECOND SPHERE

HISTIDINE IN α-AMINO-β-CATBOXYMUCONATE-ε-SEMIALDEHYDE

DECARBOXYLASE ...... 82

5.1 Introduction ...... 83

5.2 Experimental procedures ...... 85

5.3 Results 89

5.3.1 Biochemical properties of the His228 mutants ...... 89

5.3.2 The blue color in H228Y is not due to formation of a metal-bound

dihydroxyphenylalanine...... 91

5.3.3 Resonance Raman characterization suggests an Fe(III)-tyrosinate chromophore in as-

isolated Fe-H228Y...... 92

5.3.4 Spectroscopic characterization of Co(II)-substituted H228Y...... 97

5.3.5 X-ray crystal structure ...... 99

5.4 Discussion...... 104

5.4.1 The origin of blue color in H228Y ...... 105

5.4.2 The role of His228 in maintaining the hydrolytic water ligand for catalysis ...... 105

6 SUMMARY ...... 109

REFERENCES ...... 110

LIST OF TABLES

Table 1 Mössbauer parameters of as-isolated ADO and cysteamine bounded ES complex...... 51

Table 2 Summary of EPR g-values of DNICs ...... 55

Table 3 The activity of ADO fractions eluted from size exclusion column...... 64

Table 4 Metal contents of reconstituted ADO...... 72

Table 5 The apparent parameters of ADO with different metal constant...... 72

Table 6 Metal contents of copper reconstituted enzymes...... 79

Table 7 The kinetic parameters table of hCDO and hADO versus three substrates ...... 80

Table 8 Resonance Raman vibrations and LMCT bands of nonheme iron protein and model complexes with Fe(III)-phenolate and Fe(III)-catecholate chromophores...... 96

Table 9 X-ray crystallography data collection and refinement statistics ...... 100

LIST OF FIGURES

Figure 1 The overall structure of TflA (A) and the its active site (B) with substrate bond complex (C). (PDB: 3PKW, 3PKV and 3PKX) ...... 3

Figure 2 The reaction catalyzed by TflA (adopted from ref 5)...... 3

Figure 3 The function of extradiol and intradiol dioxygenase...... 5

Figure 4 The overall structure of HPCD and MndD...... 6

Figure 5 The crystal structure of 3, 4-PCD...... 8

Figure 6 Crystal structure view of TauD...... 11

Figure 7 The chemical reaction that TauD catalyzes...... 11

Figure 8 The crystal structure of IPNS...... 13

Figure 9 The crystal structures of QDO that binds copper and iron...... 16

Figure 10 The reactions that Ni/CoARD and FeARD catalyze...... 17

Figure 11 The structure of ARD with Ni (A) and Fe bounded (B). (PDB: 1VR3, 2HJI) . 17

Figure 12 The crystal structure of GDO...... 20

Figure 13 The reaction that PET catalyzes...... 23

Figure 14 Crystal structures of the active site of PTE (A) bounded with two inhibitors (B,

C)...... 24

Figure 15 Crystal structure of ADA. A overal structure (A) and the active site bounded with two substrate homologues. (PDB: 1ADD, 2ADA)...... 25

Figure 16 The reaction that LigI catalyzes...... 25

Figure 17 Crystal structure of LigI. The active site of native protein (B) and D248A mutant with substrate (C) and product bounded (D). (PDB: 4D8L, 4DI8) ...... 26

Figure 18 The reaction that Fe or Mn SOD catalyzes...... 28

xii

Figure 19 The active site of FeSOD, MnSOD and Fe substituted MnSOD...... 29

Figure 20 The reaction of Cu/Zn SOD with superoxide...... 30

Figure 21 The crystal structure of Cu/ZnSOD...... 31

Figure 22 The crystal structure of SOD (PDB 2JI3)...... 32

Figure 23 The proposed mechanism for SOR. (Adopted from ref 86) ...... 33

Figure 24 Mössbauer spectra (4.2 K; 50 mT applied field parallel to γ-radiation) of 57Fe-

enriched ADO...... 51

Figure 25 The selected ion monitoring LC-MS spectra...... 53

Figure 26 EPR spectra of ADO nitrosyl complex...... 56

Figure 27 The UV-Vis absorbance of ADO (a); ADO-NO (b) and ADO-NO-S (c) ...... 56

Figure 28 Size exclusion purification profile of as-isolated ADO with and without DTT

in the elusion buffer and the SDS results...... 63

Figure 29 The gel filtration profile of ADO with/out DTNB incubation...... 64

Figure 30 The CD spectra of ADO...... 65

Figure 31 The summary of the role of cysteine residues in ADO...... 66

Figure 32 Micaleas-Menten curve of metal reconstituted ADO...... 72

Figure 33 The ESI spectrum of CuADO with cysteamine mixture after 30 min incubation.

...... 74

Figure 34 The ESI spectrum of Cu 2+ with cysteamine mixture after 30 min incubation. 74

Figure 35 The DEPC modification reaction on histidine...... 75

Figure 36 The experiment design of using DEPC histidine modifying reagent...... 75

Figure 37 The illustration of DEPC modification experiment...... 76

Figure 38 The activity of Fe and CuADO after DEPC treatment...... 77

xiii

Figure 39 The proposed catalytic role of His228 in ACMSD reaction ...... 85

Figure 40 The active site of ACMSD...... 90

Figure 41 UV–vis spectra of Fe-H228Y...... 91

Figure 42 Resonance Raman spectra of ACMSD...... 95

Figure 43 The UV-Vis and EPR spectra of Co(II)-substituted ACMSD...... 98

Figure 44 Superimposed overall structure ...... 101

Figure 45 The active site structure of ACMSD and H228X mutants...... 103

1 INTRODUCTION

Enzymes are proteins act as catalysis, which are produced in all living cells.1 Enzymes have versatile functions that are closed related to global environment, energy production, industry applications, medicine pharmaceutical research and our daily life. Enzymes are involved in many basic life processes such as photosynthesis in plants, cell respiration, DNA/RNA replica- tion and cell division etc. A lot of diseases are caused by the lack or mutation of enzymes. The first enzyme was discovered from the sugar fermentation to alcohol by yeast and thus alcohol production is the first beneficial industry application of enzyme. The chemistry reactions that enzymes catalyze are mostly nonenzymatic energy unfavorable reactions such as C-C, C-H bond cleavage, oxygen insertion, peptide or nucleotide formation etc. 2

A considerable amount of the enzymes require binding of cofactors such as metal ion(s), flavin, NAD, biotin etc. to perform catalytic functions. Enzymes with transition metal cofactors are named metalloenzymes. Metalloenzymes could be divided into non-heme and heme two sub-

groups based on the absence or presence of heme. Heme is an iron complex with porphyrin.

Heme in some enzymes, derived from the protein biosynthesis thus this type of heme is covalent

bonded with the protein. In other enzymes, exogenous heme interacts with protein through non-

covalent interactions between iron and protein residue(s) (His, Tyr, Cys, Met). The proximal

metal ligand from protein and the distal pocket environment play important roles on the enzyme

mechanism and the enzyme behavior. Heme enzymes always present color thus the isolation is

easier compared to the colorless enzymes in the early ages for instance the characterization and

the understanding of the heme enzymes were better than colorless enzymes. Photosystem II, oxi-

dative phosphorylase, hemoglobin, myoglobin and P450 are all well studied heme enzymes.

Non-heme metalloenzymes employ metal binding motives that derive from the protein residues

2 to bind metal (A large non-heme enzyme group utilizes Fe-S cluster to function but this type of

Fe-S containing enzymes are not the interest of this study thus will not be discussed further in this dissertation). Among those protein-derived metal ligands, His, Asp, Glu, Tyr, Cys are the most popular because firstly, they are N-, O- and S-containing residues which have high affinity to metal ion and secondly the p Ka of these residues lay in the physiological pH range. Among those most favored protein residues, His is the most popular one. Based on the number of the histidine residues involving in metal binding motif, four sub-groups of metalloenzymes will be discussed as follows.

1.1 Enzymes with one histidine metal ligand

Up to date, only one metalloenzyme utilizing 1-His-2-carboxyl metal binding motif, toxoflavin lyase (TflA), has been reported in the Protein Data Bank (PDB entries: 3PKW, 3PKV and 3PKX). Toxoflavin lyase catalyzes the degradation of a toxic compound toxoflavin utilizing

Mn(II) as the cofactor in presence of reducing agent (dithiothreitol, DTT) 3. The overall structure of toxoflavin lyase is similar to proteins that belong to the vicinal oxygen chelate (VOC) superfamily 4. VOC enzymes are divalent metal required enzymes that share two βαβββ units. 5 The function of the VOC superfamily is very diverse. The members include dioxygenase, isomerase,

α-ketoglutarate (αKG) dependent oxygenase etc 6. Due to the limited unidentified structures and

diverse metal binding motif and functions, the common mechanistic features of the VOC family

are less understood compared to other protein families.

TflA has been crystallized as apo, holo, and holo enzyme-substrate complex three different forms 4. The surprising finding in these structures is that Mn(II) is ligated by an 1-His-2- carboxlate metal-binding motif with three water molecules (Fig.1). Although the catalytic mechanism of the TflA is not well understood, the toxoflavin degradation function of TflA is largely applied in agriculture to decrease the toxicity of toxoflavin to rice.3-4 In the enzyme-substrate

3 complex crystal structure, the substrate binds to the metal through the O5 in a monodentate fashion. The product detection of this enzyme was characterized recently. Philmus et al . proposed three viable mechanisms with different intermediates and products for TflA.7 The reaction product of TflA was then purified and characterized by UV-Vis, NMR and MS combined with iso- tope labeled synthetic toxoflavin 7. It’s confirmed that, the degradation of toxoflavin is the combination of oxygenation and decarboxylation that yields the triazine (Fig. 2).

Figure 1 The overall structure of TflA (A) and the its active site (B) with substrate bond complex (C). (PDB: 3PKW, 3PKV and 3PKX)

Figure 2 The reaction catalyzed by TflA (adopted from ref 5).

1.2 Enzymes with two histidine metal ligands

According to the PDB database, a lot of enzymes utilize 2-His and one/two other residues such as carboxyl containing residues (D/E) or Tyr residues as metal binding motif. The most popular metal binding motif is 2-His-1-carboxyl that is defined as “2-His-1-carboxylate facial triad” by Dr. Lawrence Que, Jr.8 Most of the enzymes with 2-His-1-carboxylate facial triad are non-heme iron dependent enzymes and this enzyme family could be further divided into several subgroups based on the catalytic function e.g. extradiol cleaving catechol dioxygenase, rieske

4 dioxygenase, Fe/ αKG dependent enzymes, pterin dependent enzymes, and antibiotic precursor producing enzyme isopenicillin N synthase (IPNS).9 The various catalytic functions of this enzyme family are largely determined by the 2-His-1-carboxylate facial triad with three open coordination sites that allows binding of different exogenous ligands such as O 2 or other substrates.

Although 2-His-1-carboxylate facial triad was first found and defined from ferrous iron contain-

ing enzymes, the 2-His-1-carboxylate facial triad is not a specific iron binding motif. Mn(II) and

Co(II) are both found active in different enzymes and in some particular cases, one enzyme uses

other metal ions to promote the same reaction or exhibit a distinct function. The mechanisms of

the enzymes with 2-His-1-carboxylate facial triad are very well understood based on the excel-

lent study with different approaches form several groups 8, 10 .

The other protein family that utilizes 2-His as metal binding residues is intradiol ring- cleaveage dioxygenase family. Intradiol dioxygenases employ a 2-His-2-Tyr Fe(III) center for catalysis. Examples of the enzymes with different metal binding motif will be discussed as follows.

1.2.1 Ring cleavage dioxygenases—Extradiol and Intradiol dioxygenases

Extradiol/intradiol ring-cleaving catechol dioxygenases play a key role in the degradation

of aromatic compounds. Ring-cleaving dioxygenases have very broad substrate range. The sub-

strate specificity that is required by extradiol/Intradiol ring-cleaving is the ortho-orientated two

hydroxyl group on the ring structure. 11 Homoprotocatechuate 2,3-dioxygenase and protocatechuate 3, 4-dioxygenase are very well understood extradiol and intradiol dioxygenases.

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) is a charac-

teristic non-heme iron(II) extradiol cleaving catechol dioxygenase. HPCD catalyzes the cleavage

of the C2–C3 bond of homoprotocatechuate (HPCA) and the insertion of dioxygen to form the

product 5-carboxymethyl-2-hydroxymuconic semialdehyde (5-CHMSA) .12 HPCD has a homo-

5 logue protein named manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase

(MndD) that has been isolated form Arthrobacter globiformis .13 These two enzymes have similar tertiary structures and active sites with 83% of sequence identity (shown in Fig.4) 14 . Although

MndD binds Mn(II) instead of Fe(II) for HPCD, they both produce the same products from the

same substrates 15 .

se na ge xy CHO io d ol CO2H di tra Ex OH OH

+ O2 OH In tra di ol dio CO H xy 2 ge na CO H se 2

Figure 3 The function of extradiol and intradiol dioxygenase.

Swapping metals in FeHPCD and MndD doesn’t affect the catalytic activity or change

the metal redox state during oxygen activation 16 . HPCD was also found to be able to bind Co(II) to function even with higher activity than the native FeHPCD at oxygen saturating conditions 17 .

Both HPCD and MndD were crystallized as a homotetramer. 18 In each monomer, it has four βαβββ secondary structure cores suggesting they belong to VOC superfamily. The active site and the second sphere residues in both enzymes are identical thus the metal selectivity in these enzymes cannot be specified. 18

Figure 4 The overall structure of HPCD and MndD. A is the overall structure of the HPCD. B-D are the intermediates of HCPD trapped in crystalline (PDB Entry: 2IGA). E and F are the active site view of MndD with substrate HPCA (PDB 1F1R; 1F1V).

The mechanism of HPCD and MndD are very well studied from the combination of spectroscopic tools and crystallography. The intermediates were first proposed by spectroscopy and then trapped in crystal structures in wild type or mutants of HPCD. One interesting finding about

HPCD was that three intermediates were trapped in one tetrameric crystal using an alternative substrate 4-nitrocatechol (4NC) as shown in Fig. 4ABCD.14a The first intermediate is a side-on

O2-Fe superoxide. The next intermediate shows that the O 2 has attacked the substrate to yield a

substrate-O2 complex named alkylperoxo intermediate, which is the key intermediate for the O 2

activation propose in the mechanism. An O 2 attached open-ring product intermediate was also

trapped in one of the four subunits. Other intermediates proposed previously based on spectro-

scopic study were approached through smart designed mutations. It was found in HPCD that

some surface residues increase the stability the intermediates observed by spectroscopy tools,

thus, one E323L mutant was constructed. This E323L mutant doesn’t affect the catalysis signifi-

cantly but the O-O cleavage intermediate is stabilized for longer life time thus the chance of be-

ing trapped in crystals is increased. Not surprising, in the crystal structure of E323L, the O-O

7 cleavage intermediate was trapped, which confirms that in the HPCD catalysis the two oxygen atoms are inserted to the substrate sequentially instead of spontaneously.19

The function of the second sphere residues was also thoroughly studied. A highly conserved His200 was found to play a dual role as active acid-base and H-bonding donor to the enzyme-substrate-dioxygen ternary complex. 14b Another hydrogen bond donor to the dioxygen is

Trp192. The mutants of the His200 in HPCD results in very low enzyme activity, interestingly, the H200F FeHPCD mutant was found to have both extradiol and intradiol dioxygenase activity. 20 A conserved active residue Tyr257 was found to distort the substrate through hydrogen bonding and van der Waals interaction with C2 on the substrate for radical generation and alkylperoxo intermediate formation. 21

A consensus mechanism for HPCD has been proposed based on spectroscopic and crys-

10a, 14a, 21-22 tallographic results. The unique features of the HPCD are that: 1) O 2 activation is

through primary substrate radical intermediate; 2) metal oxidation state remains the same during

the catalysis; 3) O-O bond cleavage is required for product formation and 4) second sphere resi-

dues play important roles on the catalytic mechanism.

HPCD is the best example to address the interest of this dissertation that lies in the metal

binding motif specificity, the metal selectivity and the function of the second sphere residues.

Several intradiol dioxygenases have been characterized 23 . Protocatechuate 3, 4-

dioxygenase (3, 4-PCD) that catalyzes the oxidation of protocatechuic acide to 3-carboxy-cis ,

cis-muconate by breaking C3-C4 bond will be discussed as a representative model. 3, 4-PCD

was isolated in 1968, which was reported as a Fe(III) dependent non-heme enzyme 24 . Due to its

high protein yield, easy crystallization conditions and the spectroscopic features, 3, 4-PCD was

very well characterized afterwards. The iron center in 3, 4-PCD is ligated by a 2-His-2-Tyr metal

binding motif plus a hydroxide ion in distorted bipyramidal geometry. Among the five metal lig-

8 ands, His462, Tyr408 and the hydroxide ion are equatorially bound to the metal center while

Tyr447 and His460 are the axial ligands. Tyr447 was observed to be replaced by the substrate in the substrate-enzyme complex crystal structure and affect the product release.19c, 23c, 25 20c, 21 This

2-His-2-Tyr metal binding motif-containing dioxygenase has not been found active with other metals.26

The loss of the one equatorial ligand Tyr408 does not affect the metal binding but resulted in inactive enzyme. 27 The crystal structure of Tyr408 mutants with PCA complexes showed that other residues are not able to ligate the iron and PCA bond the metal monodentately thus

Tyr447 failed to dissociate from the iron center. The function of Tyr408 is more on the substrate binding and activation other than the metal center itself. The substrate activation is also correlated closely with the substrate’s chemical feature. The substitution of 4 -nitrocatechol (4-NC) with the native substrate PCA inhibits the enzyme activity although the binding of the two are very similar in the crystal structure of 4-NC/PCA with enzyme complexes.23c

Figure 5 The crystal structure of 3, 4-PCD.

9 A The overall structure (PDB 2PCD); B The active site view of the enzyme (PDB 2PCD); C The active site view of enzyme bond with 4-NC (PDB 1EOC); D The active site view of the enzyme with native substrate (PDB 1EOB); E The active site view of the enzyme bond with INO (PDB 3PCJ) and F in presence CN (3PCL).

The difference between the two complex structures is that the three well-ordered water molecules that are hydrogen bonded with the carboxyl group with PCA are missing in the 4-NC-

enzyme complex and therefore the 4-NC could not be activated after binding. The O 2 activation

was thought to be through the substrate ligation that is because Fe(III) is not an ideal O2 ligand

plus the crystal structure of enzyme-substrate complex showed the dissociation of the Tyr447

28 opens a small cavity adjacent to the C3-C4 for O 2 binding. In the proposed mechanisms, the

iron center doesn’t change the oxidation state. The spectroscopic investigation and computational

calculation of the metal center after binding of substrate suggested that a very strong π interac-

tion formed between the substrate and Fe yielding the lowest energy PCA-to-Fe(III) LMCT ( lig- and-to-metal charge transfer) transition thus the Fe(III) in 3, 4-PCD functions like a buffer for the spin-forbidden two-electron redox process between PCA and O 2 in the formation of the en-

25 zyme-substrate-O2 complex.

HPCD and 3, 4-PCD share a similar reaction mechanism; however in 3. 4-PCD, the Tyr

408 in the 2-His-2Tyr binding motif plays a dual role. It binds to metal in the resting state enzyme but plays a role of substrate stabilization and activation when flapping away from the metal after substrate binding. This is the first example reported in enzyme that the metal binding ligand acts as a second sphere residue during catalytic reaction.

1.2.2 Fe/ α- ketoglutarate( αKG) dependent enzymes

Fe/ αKG-dependent enzymes a group of versatile enzymes function in protein posttrans-

lational modification, DNA/RNA repair , biosynthesis of antibiotics, plant products synthesis,

lipid metabolism and biodegradation of a variety of compounds.29 Fe/ αKG-dependent enzymes

10 share a structure “ β-sheet jelly roll” feature thus they belong to the cupin superfamily. The metal binding coordination in this group of enzymes is the typical 2-His-1-carboxylate facial triad.

Fe/ αKG-dependent enzymes require the binding of αKG to initiate the reactions. Although Fe/

αKG dependent enzymes are sometimes referred to as Fe/ αKG-dependent dioxygenase, strictly speaking they are hydroxylases because the two oxygen atoms are inserted to two different substrates: one is incorporated into the αKG decarboxylation production succinate while the other is inserted into the final product of the primary substrate through high-valent iron species.

The first direct characterized high-valent iron species in Fe/ αKG-dependent hydroxylases is TauD. TauD belongs to the cupin superfamily with a single cupin domain (Fig. 6A). The overall reaction that TauD catalyzes is shown in Fig. 6. The proposed mechanism of TauD has three distinct features compared to ring-cleaving dioxygenase: 1) the oxygen activation through Fe(II) after αKG binding; 2) O-O bond cleavage is through the formation of oxo-ferryl coupled with decarboxylation of the αKG and 3) the second oxygen is inserted to the primary substrate C-H bond by the oxo-ferryl compound through substrate radical intermediate 30 .

Unlike the metal promiscuous feature of extradiol cleaving catechol dioxygenase’, αKG - dependent hydroxylases only prefer to bind iron (II) to function. Binding of other metal ions results in the inactivation/inhibition of the enzyme.31 The metal selectivity in Fe/ αKG-dependent enzymes is mainly determined by the reaction requirements of high-valent species formation.

Figure 6 Crystal structure view of TauD. A The overall structure of TauD; B The active site of Enzyme-αKG-Taurine complex (PDB Entry: 1GQW).

Figure 7 The chemical reaction that TauD catalyzes.

In the co-crystal structure of TauD-αKG-taurine complex, the primary substrate taurine does not bind to the iron center but it is very well positioned by forming multiple hydrogen bonds with rounded resides through both nitrogen and oxygen atoms. The C-H bond that needs to be broken is right on top of the Fe center. This feature significantly narrows the substrate specificity. The co-substrate αKG is bidentately ligated to the iron center, which is also stabilized by forming charge pairs with R266 (Fig. 7). TauD is the first found dioxygenase that insert oxygen to the primary substrate by ferry-oxo species.

12 1.2.3 Antibiotic biosynthesis related enzyme- Isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyzes a four-electron oxidation reaction in the biosynthesis of penicillin cephalosporins and cephamycins.32 Before the first crystal structure came out, an EPR spectroscopic study showed that IPNS is a non-heme iron enzyme.33 The first crystal structure of IPNS shows a dimmer form of Mn-containing metal center that is ligated with 2-His-

1-carboxylate facial triad. The overall structure of IPNS presents a “Jelly-roll” showing that

IPNS is one of the cupin family members.10b

Mechanistically, IPNS is different from the above introduced two enzyme families although they share similar structural features. IPNS doesn’t insert oxygen into the its substrate ( δ-

(L-α-aminoadipoyl)-L-cysteinyl-D-valine , ACV), instead, it draws four electrons from the substrate and kicks out four protons to dioxygen forming two water molecules. At the same time, two rings are closed on ACV stepwise to generate the produce Isopenicillin N. The previous spectroscopic study suggested that ACV binds to the ferrous iron center through the substrate thiolate and oxo-ferryl is formed. 33-34 The key intermediates during the two ring closure were trapped stepwise in crystalline using synthesized substrate homologues that has lower activity. 35

Fig. 8 shows the overall structure of IPNS and the active site structure in complex with its substrate, one ring intermediate and product ligation. The electron transfer is through the sulfur-iron bridge. Three secondary residues Ser281, Tyr189 and Arg89 stabilize the substrate during the catalysis through either hydrogen bonding or charge pair electrostatic interaction.

Figure 8 The crystal structure of IPNS. A The overall structure; B The active site of ES complex; C The active site of one ring closure product and D The product formation in crystal (PDB Entry: 1QJE, 1QJF)

In summary, extradiol ring cleave dioxygenases, Fe/ αKG -dependent enzymes and an antibiotic biosynthesis related enzyme with unique reduction products Isopenicillin N synthase are highlighted. The three enzymes that picked up as examples from each sub-group are well studied and understood. The advantages of 2-His-1-carboxylate facial triad are: 1) it opens three more coordination sites for exogenous compounds to bind and this is why the enzymes in this family are versatile; 2) with the binding of metal, the active site is in charge balance so that there is no limit for substrate selection and that is also the second reason why the enzymes catalyze numerous reactions. The 2-His-1-carboxylate facial triad is quite a metal promiscuous motif; however, the function of enzyme is not only determined by metal but also determined by other factors such as the second sphere residues and/or the electronic statistics of the whole protein.

14 1.3 Enzymes with three histidine metal ligands

Three are two major protein families that use three histidine residues in the metal binding

motif: one sub-group of cupin superfamily and amidohydrolase superfamily.

Cupin is derived from a Latin world “cupa” meaning small barrel. Proteins in cupin su-

per-family are known to have a common β-barrel structure and also known as a “jelly roll” bar-

rel 36 . The metal binding motif are identified as cupin metal binding motif that contains character-

37 istic conserved tow short sequences of G(X) 5HXH(X) 4E(X) 7G and G(X) 5PXG(X) 2H(X) 3N .

Most of the proteins in the cupin superfamily have highly conserved 3-His-1-catboxylate or 2-

His-1-carboxylate metal ligands but a small portion of the members lost the carboxylate residue

during revolution 38 . The di-valent transient metals such as iron, copper, nickel, cobalt and manganese have all been found in cupin proteins. 39 The function of the cupin members includes dioxygenation, decarboxylation and gene regulation etc.40

Amidohydrolase superfamily is a huge protein family that catalyzes the hydrolysis of a wide range of the substrates, deamination of nucleic acids, decarboxylation, isomerization, and hydration 41 . The featured structural motif of amidohydrolase is the 8 α8β TIM barrel. The metals

that have been found in enzymes from amidohydrolase superfamily are Zn(II), Ni(II), Fe, and

Mn.41a, 42 Some of the members bind a bi-metal core, some only bind one metal ion and one newly discovered member is found to bind no metal but uses the same metal bind ligands to function.43 Some well understood enzymes from each protein super family will be discussed as

representative examples.

1.3.1 Dioxygenases with 3-His-1-carboxylate motif in cupin superfamily

Cupin superfamily members could be divided into two large subgroups based the func-

tion: enzymes and non-enzymatic transcription factors that have a conserved C-terminal domain

that interacts with DNA.40d

15 1.3.1.1 Quercetin 2,3-dioxygenase (QDO)

Quercetin 2,3-dioxygenase (QDO) has been isolated from Aspergillus flavus (CuQDO) and Ba- cillus subtilis (FeQDO) with coordination of the copper and iron, respectively 44 . QDO has two cupin domains thus it belongs to the bicupin sub-superfamily. The copper center in QDO has two geometries: distorted tetrahedral coordination that has three histidine resides (His67, His68 and

His112) plus one water molecules or distorted trigonal bipyramidal coordination that has one additional ligation from Glu73.45 CuQDO is the first found dioxygenase that utilizes copper as metal center. The reaction that QDO catalyzes is dioxygen insertion to the substrate quercetin coupled with carbon oxide releasing 46 . The metal substitution study of Fe(II) containing FeQDO showed that FeQDO is active with substitution of Fe(III), Cu(II), Mn(II), Co(II), Ni(II). The surprising finding of the metal study is that Co(II)- and Cu(II)-reconstituted FeQDO proteins are about two folds more active than the native ferrous iron while zinc-reconstituted protein is inactive. In the CuQDO case, there is no reported activity for other metals.47 As shown in Figure 9, one monomer of QDO has two cupin domains but one cupin domain has the active site. Two active geometries have been observed in CuQDO upon the binding of Glu73 to metal or not (Fig.

9B). In FeQDO, only one type of geometries of the active site has been observed (Fig. 9C). The

CuQDO enzyme-substrate soaking complex structure shows the monodentate interaction of

quercetin (QUE) and kaempferol (KMP) (Fig. 9D, E). The bindentate chelation of substrate

homologue kills the enzyme.

The FeQDO and CuQDO may have different oxygen activation mechanisms. In FeQDO,

dioxygen binds to the Fe(II) center forming Fe(III) superoxide but in CuQDO O 2 is not a good ligand to the Cu(II) ion. Thus, the dioxygen activation in CuQDO is more likely through the substrate radical mechanism. One reasonable propose is that Cu(II)-substrate binary complex is in equilibrium with Cu(I)-substrate radical through tautomerization that opens two dioxygen bind-

16 ing spots on both Cu(I) site and the substrate radical site 46 . So the function of the metal center in

CuQDO is a buffer function like the Fe in 3, 4 PCD intradiol dioxygenase than being an oxygen activation pool. A similar concept is raised in intradiol dioxygenase. 48 FeQDO, however is thought to use a different dioxygen mechanism that is similar to extradiol catechol dioxygenases. 49

Figure 9 The crystal structures of QDO that binds copper and iron. A The overall structure of QDO. B The active site of CuQDO and FeQDO. (C). D-F The CuQDO complexes with substrates or inhibitors. (PDB: 1JUH, 1Y3T, 1H1I, 1H1M, 1GQH ).

1.3.1.2 Acireductone dioxygenase (ARD)

Acireductone dioxygenase (ARD) was first discovered during the studies of the methionine salvage pathway in Klebsiella pneumonia, which catalyzes the oxidative degradation of the acireductone to formate, CO, and methylthiopropionic acid with the consumption of dioxygen.50

17 O- S O- O CO O NiARD

S OH OH

FeARD OH S O- O O

Figure 10 The reactions that Ni/CoARD and FeARD catalyze.

Different structures of ARD have been obtained by NMR,51 X-ray absorption spectroscopy,52

residual dipolar couplings,53 as well as X-ray crystallography.54 ARD was found to contain one cupin domain with 3-His-1carboylate metal binding motif. Fe(II), Ni(II) and Co(II) are all found active in different ARD isomers; however Fe(II) containing ARD generates different product from Ni(II)/Co(II) containing enzyme from the same substrate.55 Even though Fe(II) or Ni(II) substitution to apo form protein both result in active form of the enzymes producing “two enzymes from one protein,56 Ni(II) or Co(II) has higher affinity to the active site compare to

Fe(II).50a

Figure 11 The structure of ARD with Ni (A) and Fe bounded (B). (PDB: 1VR3, 2HJI)

18 The crystal structures of Ni and Fe bounded ARD are very similar (Fig. 11). In both proposed catalytic mechanisms for ARD, the metal center retains the same oxidation state and the oxygen activation is through the substrate binding.

1.3.2 Dioxygenase with unique 3-His iron binding motif in cupin superfamily

3-His iron binding motif is relatively less popular. Thus far, the identified enzymes with

3-His metal ligands are all dioxygenases; however the catalyzing reactions and the mechanism are very different from each other. Examples of thiol dioxygenase, ketone cleaving dioxygenase and third type of ring cleaving dioxygenase will be discussed.

1.3.2.1 Gentisate 1, 2-dioxygenase

Gentisate 1, 2-dioxygenase (GDO) is a different type of ring cleavage dioxygenase compared to extradiol/intradiol dioxygenases that demand substrates with two hydroxyl groups in ortho -orientation to each other. GDO and the homologues enzymes catalyze the cleavage of C-C bond, the carbon of which are substituted with carboxyl and hydroxyl group, respectively57 .

The first crystal structure of GDO from Escherichia coli named (eGDO) revealed that

GDO is a bicupin protein with one 3-His iron binding motif at the N-terminal domain.58 Interest- ingly, one of the GDO homologues form silicibacter pomeroyi has two iron centers with 3-His metal binding motif located in both N and C cupin domains. The kinetics study of N or C terminal truncated enzyme showed that the presence of both iron is required for the enzymatic activity. Although the mechanism of the binuclear GDO is still unknown, the comparison of the crystal structure with homologues together with the various mutation studies suggested that the iron center at the N-terminal is the active center which has long distance cooperation with the other iron center in C-terminal domain.59

The biochemical and structural characterization of GDO show that it is an iron dependent enzyme that is inactive with other divalent metals, however, GDO is a substrate promiscuous en-

19 zyme that could catalyze the oxidation of several ring containing compounds with ortho - orientation carboxyl group and hydroxyl groups.58, 60 The GDO homologue from bacterium

Pseudaminobacter salicylatoxidans BN12, which was named as salicylate dioxygenase (SDO) oxidizes a broad range of substrates including gentisate, 1-hydroxy-2-naphthoate (1H2NC) and salicylate(s).61 SDO was crystallized as a tetramer. The co-crystal structure of SDO with salicylate, gentisate and 1H2NC revealed the bidentate binding manner of the substrates to the metal center and several second sphere residues, which are Arg127, His162, and Arg83 in specific, form hydrogen bonds with the carboxyl group on the substrate. The movement of loops composed of residues 40–43, 75–85, and 192–198 were was also observed upon substrate binding 57 .

It is notable that the binding of different substrates in the co-crystal structures altered the location of several second sphere residues like Trp104 and Arg83 that interact with the substrates through electrostatic or hydrogen bonding interactions. The substrate specificity of SDO toward gentisate versus 1H2NC was studied through mutation of several second sphere residues and was nar- rowed on one residue Ala85. A85H was found to only catalyze the oxidation of 1H2NC but completely lost the activity toward gentisate. The crystal structure of A85H showed that the bulky side chain of histidine occupies some space of the active site. The structure difference between gentisate and1H2NC allows the 1H2NC to binding to the metal but not gentisate.61 The

W104Y mutant shows a reduced activity on all three substrates indicating that Trp104 is not related to the substrate specificity.

In summary, GDO is a metal strict iron dependent dioxygenase on various substrates that have ortho -orientation carboxyl group and hydroxyl groups. The second sphere residues play important role on the mechanism and substrate selectivity through hydrogen bounding or electrostatic interaction with substrate.

Figure 12 The crystal structure of GDO. A The overall structure of GDO is a tetramer (PDB: 2D40). B The active site of the GDO (PDB: 3NST). C The salicylated bounded GDO (PDB: 3NJZ) D The 1H2NC bounded GDO (PDB: 3NKT). E Gentisate bounded GDO (PDB: 3NL1). F The gentisate bounded W104Y mutant (PDB: 4FAG).

1.3.2.2 Thiol dioxygenase

Cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO) are the only reported thiol dioxygenases in mammal so far. Both of the enzymes belong to cupin superfamily. The reactions that the two enzymes catalyze are the oxidation of the free thiol on cysteine and cysteamine via dioxygen insertion. These two enzymes play very important role on the cysteine metabolism, redox regulation and neurotransmitter generation. The substrate of ADO cysteamine is widely used in the biochemical and pharmaceutical applications.

More than 50 years ago, CDO was found to convert L-cysteine to L-cysteine sulfinic acid 62 . In 2006, Simmons et al. over expressed and purified CDO in vitro , which busted the structural and mechanistic study of this enzyme. 63 The crystals structure of CDO cloned from rat 64 , mouse 65 and human 66 were all solved in the past few years. In all the three CDO structures,

21 the metal ion is ligated to 3-Histidine protein residues (2B5H, 2ATF, 2IC1) revealing a unique metal binding motif in cupin superfamily for the first time. In 2007, a hypothetical murine protein homolog of CDO, which is encoded by the gene Gm237 in DUF1637, was identified to have significant cysteamine dioxygenase activity in vitro 67 . This protein homology of CDO was named cysteamine dioxygenase (ADO). The sequence alignment suggests that ADO not only belongs to the cupin superfamily but also has a 3-His metal binding motif as CDO in despite the sequence similarity of the two proteins is only about 14%-19%. Upon its first isolation from horse kidney in 1963.68 ADO was reported to require sulfide, elemental sulfur, selenium or hydroxylamine to function. 69 The recombinant ADO was reported to use ferrous iron as a cofactor to catalyze requiring none of the exogenous elements mentioned above.67 Still, information about the structure and mechanism remain sparse.

Several mechanisms for CDO have been proposed. The difference among them lies in several key questions: 1) how is dioxygen activated; 2) if oxo-ferryl is required and 3) if O-O bond cleavage before oxygen insertion is necessary. Oxygen activation could be via two different ways: through metal ligation most of the times forming superoxide reactive species or substrate ligation through substrate radical mechanism. In the typical examples that have given above, the reactions are more or less very “difficult reactions” because the oxygen insertion needs to break

C-C bond or C-H bond. Here in CDO, the substrate biochemical feature is very different from others, which is an electron rich compound. The oxygen insertion happens on one sulfur atom, which does not require any bond cleavage in the substrate, thus the thiol dioxygenase should have a different reaction mechanism. The difficulties of the studying the mechanism for both

CDO and ADO is that neither the protein itself nor the substrate has visible chromospheres the change of which could be detected and followed by UV-Vis absorption. Fortunately, CDO could be crystallized and a ES ternary complex was trapped in crystalline; however, no other interme-

22 diates proposed in the mechanism have been trapped yet thus the mechanism for CDO is still un-

der investigation. Even Ni(II) is bounded in the first published CDO crystal structure, CDO is

reported as a Fe(II)-dependent dioxygenase.

Unlike to CDO, there is not much biochemical characterization or crystallography report

on ADO thus far.

1.3.2.3 Diketone-cleaving dioxygenase (Dke1)

Dke1 is one of the few protein 3-His metal binding motif-containing enzymes with re-

ported crystal structures (PDB: 3BAL). Dke1 catalyzes the oxidation of 2,4-pentanedione in

presence of O2 into methylglyoxal and acetate. In the reported crystal structure, zinc is substituted to iron. DKe1 is multifunctional enzyme. The native Fe(II) enzyme catalyze both β-Diketone- cleavage and 4-nitrophenylester hydrolysis while Zn(II) substituted enzyme is only able to function as hydrolyse.70

The mechanism is approached by spectroscopic and computational studies. The oxygen activation is through substrate ligation. Ferrous iron retains the same oxidation all the through the reaction.71 The second shell residues are more hydrophilic, which are responsible for metal

72 stabilization, substrate binding and O 2 reduction.

The 3-His iron binding motif derives from 3-His-carboylate in cupin superfamily by re- placing the carboxylate group with non-conserved residues. 3-His-1-carboylate dioxygenases is a metal binding promiscuous motif with a little higher preference for ferrous iron; however, the dioxygen activity is not determined by the metal selectivity. The function of the 3-His is unclear thus far.

1.3.3 3-His-1-carboxylate containing enzymes amidohydrolase superfamily

Amidohydrolase could be divided into eight subgroups based on the ligands that involved in the metal binding. The metal(s) center in the amidohydrolase plays a key role of the

23 deprotonation metal ligated water to form nucleophilic attack hydroxyl group.41a Enzyme that

have mononuclear 3-His-1 carboxylate metal binding motif will be given as examples.

1.3.3.1 Binuclear metal containing enzyme phosphotriesterase (PTE)

PTE is one of the first identified amidohydrolase superfamily members by Holm and

Sander.73 PTE catalyzes the hydrolysis of a phosphorus-oxygen bond within an organophosphate

trimester. Two zinc centers are found in this protein with about 3.4 Å away from each other. One

zinc center is ligated to the protein fold through 2-His-1-Asp residues while the other is by 2-His

residues. In between the two zinc metal ions, a carboxlyated Lys and one water molecule bridge

the binuclear metal core. The metal switching study showed that this binuclear metal binding

motif could also bind Zn(II)/Cd(II), Cd(II)/ Cd(II), Mn(II)/Mn(II) and Zn(II)/Cd(II) to function 74 .

The two metal ions in PTE have different functions in the catalysis. The more buried metal ion

(M α) with 2-His-1-Asp coordination binds to the carbonyl and phosphoryl groups of the sub-

strates to polarize them through Lewis acid catalysis. The other more solvent exposed metal ion

(M β) is responsible for the metal-bind water deprotonation for nucleophilic attack while the hydrolytic water molecule is activated for nucleophilic attack.41a

Figure 13 The reaction that PET catalyzes. 1.3.3.2 Adenosine deaminase (ADA)

Adenosine deaminase (ADA) catalyzes the deamination of adenosine to form inosine in the pu- rine savage pathway. 75 The crystal structure of the ADA shows that it is a monomer with 3-His-1 carboxylate ligated mononuclear zinc containing enzyme. 76

24 The reaction mechanism of the ADA is a typical mechanism that shared by amidohydrolase superfamily members. Metal switch study in ADA shows that only Zn(II) is active, however, in a homologue protein cytosine deaminase (CDA) that coverts cytosine to uracil requires Fe(II) as the metal cofactor. 77 Other metals are either inactive or inhibitors to both of the two proteins. In the crystal structure of the ADA (Fig.12), one metal ligated water molecule is stabilized by the second sphere residue His238 that deprotonates the water once substrate is bounded. The substrate activation is through the deprotonation of the substrate nitrogen by Glu217. The inhibitor and ADA complex active site shows that the substrate is monodentately ligated to the metal center.

ADA is a very typical amidohydrolase that activates the substrate through proton transfer and generates the hydroxide nucleophile by a second sphere general base. 41a

Figure 14 Crystal structures of the active site of PTE (A) bounded with two inhibitors (B, C). (PDB: 1HZY,1DPM, 1EZ2)

Figure 15 Crystal structure of ADA. A overal structure (A) and the active site bounded with two substrate homologues. (PDB: 1ADD, 2ADA). 1.3.3.3 2-Pyrone-4,6-dicarboxylate lactonase (LigI)

LigI catalyze the hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate

and 4-carboxy-2-hydroxymuconate in the degradation of lignin. LigI is a member of

amidohydrolase superfamily; however, it doesn’t bind metal ion as a cofactor to function 43 . The observations from the crystal structure suggest that the substrate activation is through the ion pairs with two Arg residues while the water molecular is deprotonated by a nearby Asp residue.

LigI thus is a new member in amidohydrolase superfamily with distinct structure and function features.

O O O- O- HO O LigI O -O -O O O O HO

Figure 16 The reaction that LigI catalyzes.

As shown in the crystal structure of LigI (Figure 14B), the native protein active sit is composed

of 3-His-1 carboxyl residues but without metal ligation. The D248A mutation allowed the ES

intermediate trapping in crystallo as shown in Fig.17C. The substrate is an oxygen rich com-

pound thus could be stabilized either by hydrogen bounding (marked by blue dashes) or electro-

static interactions (marked by orange dashes). The product has more interactions with protein

26 residues as shown in Fig. 17D. Thus far, there are no metal analysis has been done on this enzyme to confirm the metal content in the protein; however, the addition of the exogenous metal ions doesn’t affect the catalytic efficiency of this enzyme. The mutations of the active site resides that interact with substrate resulted in dead enzyme. The mechanism of the LigI was proposed to be initiated by the water deprotonation by Asp248 that generates the hydroxyl anion to attach the

C-2 carbonyl group of the substrate.

Figure 17 Crystal structure of LigI. The active site of native protein (B) and D248A mutant with substrate (C) and product bounded (D). (PDB: 4D8L, 4DI8)

LigI represents a new sub-group in amidohydrolase superfamily, the catalytic ability of which is not metal dependent. Interestingly, the metal binding residues are conserved in LigI but just not metal ligation. The active site histidines and caryboxyl are responsible for substrate activation and active site water deprotonation.

1.3.4 Α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD)

ACMSD was firstly purified from hog kidney cytosol by a series of purification steps. 78

The decarboxylation of α-amino-β-carboxymuconate-ε-semialdehyde is a key step of niacin syn-

27 thesis from tryptophan in mammals and 2-nitrobenzoic acid biodegradation in bacteria 78-79 . Alt-

hough the physiological function of ACMSD was studied via the animal model, the function or

the structure of the enzyme remained as a mystery for a very long time.80 The kinetic and spectroscopic study of ACMSD shows that ACMSD is a metal ion required decarboxylase that pur- sues nonoxdidative decarboxylation. It is inactive after EDTA treatment but active with Co(II),

Fe(II), Cd(II) and Mn(II) substitution to apo enzyme. Other metals like Fe(III) or Cu(II), Ni(II),

Ca(II) and Mg(II) are not active.79 ACMSD from Pseudomonas fluorescens was crystallized as a dimer with a ( β/α)8 TIM barrel central core and single metal ion in each monomer. The X-ray fluorescence scan of native protein crystals reveals that ACMSD is a Zn(II)-dependent decarboxylase in amidohydrolase superfamily. The zinc ion in ACMSD is ligated by 3-His-1- carboxylate plus a water molecule. 81 Two mechanisms were proposed based on the crystal structure, which metal-bounded water is activated by a nearby active site base.82 The pre-steady kinetics study of ACMSD shows two intermediates during the react which are proposed as the product α-aminomuconate-ε-semialdehyde (AMS) complexed with (intermediate I) and release from

ACMSD (intermediate II).

ACMSD is a unique member in amidohydrolase superfamily, which catalyzes the decarboxylation reaction instead of the substrate hydrolysis; however, the overall structure and active site similarity suggests that the two complete different reactions may have similar mechanisms.

It’s known that in amidohydrolase enzymes, the reaction mechanism is initiated by the deprotonation of the active site water molecule by the metal center and/or a nearby amino acid residue and the same time the substrate activation is via the binding to metal center or through the hydrogen interactions with the nearby amino acid residues. In ACMSD, a water molecule is ligated with metal center, nearby residues such as Arg51, His228 are candidates corresponding to substrate or water activation. The two product-related intermediates trapped in the pre-steady

28 study suggest the product releasing the rate limiting step of the whole reaction. Unfortunately, the reaction of ACMSD is too fast to yield a long-live intermediate during the decarboxylation such that the mechanism of ACMSD is still unrevealed.

1.3.5 Superoxide dismutase with 3-His-1carboxylate motif

Superoxide dismutase (SOD) is a group of enzymes that convert the super oxide anion radical to hydrogen peroxide and oxygen. There are three types of the SOD have been studied: binuclear Cu/Zn SOD, mononuclear Ni containing SOD and mononuclear Fe or MnSOD. 83 The

Fe or Mn SOD are homologue proteins that have very similar protein folding with 43% amino acid identity and active site metal geometry, which share very low homology to Cu/Zn SOD or

NiSOD. The 3-His-1-carboxylate metal binding motif is highly conserved in both FeSOD and

MnSOD. The reaction that Fe and Mn SOD catalyze is a coupled reaction of the oxidizing and reducing superoxide to oxygen or hydrogen peroxide in one turn over reaction.

M(III) O2 M(II) O2

M(II) O2 2H M(III) H2O2

Figure 18 The reaction that Fe or Mn SOD catalyzes.

Fe and Mn SOD have similar catalytic behaviors such as their activity both decrease at high pH and they are both inhibited by azide or fluoride anion. The Fe substitution to MnSOD doesn’t alter the active site as shown in Fig. 19 Band C; however, unlike QDO or HPCD that have been introduced above, metal swapping of the two SODs resulted in dead enzymes.

Figure 19 The active site of FeSOD, MnSOD and Fe substituted MnSOD. (PDB: 1ISA, 1VEW and 1MMM)

The proposal of the redox potential difference between the Fe and MnSOD is proved to be true.

The redox potential of Fe-substituted MnSOD is much lower compared to FeSOD that it only reduces the superoxide to hydrogen peroxide. 84 Mn-substitution to FeSOD increased the redox potential dramatically,

thus lost the catalytic activity. The second sphere residues in the active site of the two enzymes are highly conserved. Trp126, Glu80, and Tyr34 are universal conserved. Gln146 in

MnSOD positions the same as Gln69 in FeSOD. The redox tuning in Fe or MnSOD is through hydrogen bond mediation of those second sphere residues, which is directly mediated through the p K values of the active site. 85 The study mutations of the essential second sphere residues

Gln69, Trp123 and Tyr34 revealed that those two residues are involved in the catalysis by providing hydrogen bonding interactions to the active site water. 86

SOD is a special enzyme that catalyzes the reaction on a very reactive substrate. One turnover of the catalysis requires two equivalent of the substrate preceding two half reactions with different products. Due to the features of the reaction, SOD is designed to employ a metal center with specific surrounding second sphere residues that do not react with the substrate but provide perfect tuning function for the metal at different redox state to be able to oxide or reduce the substrate in one turnover. It also because of this precise redox feature requirement of the metal in SOD, the enzyme is metal conserved.

30 1.4 Enzymes with four histidine metal ligands

Enzymes with 4- His ligands are rare. There are two reasons for the rareness of 4-His metal binding ligand containing enzymes: 1) the coordination of the commonly used transition metal ions in metalloenzymes is mostly less than 6. After binding with 4-His ligands, less than 2 coordination positions are left for exogenous ligands to bind thus 4-His in less favored enzymes that require more than two exogenous substrates/cofactors. 2) 4-His plus a cationic metal ion is overall positive charged thus the compounds that could get to active pocket is limited.

Cu/Zn SOD and superoxide reductase (SOR) will be introduced as examples. 87

1.4.1 Cu/Zn binuclear SOD

The Cu/Zn containing SOD is mostly used by eukaryotes and first reported in 1975. 88

Cu/Zn SOD catalyzes the same reaction in Fig. 18 but the metal redox change is between Cu(II) and Cu(I).

Cu(II) O2 Cu(I) O2

Cu(I) O2 2H Cu(II) H2O2

Figure 20 The reaction of Cu/Zn SOD with superoxide.

The first crystal structure of Cu/ZnSOD from bovine erythrocyte showed a homodimer with one cupin fold in each monomer. The two divalent metals ions are bridged by one histidine residue

His61 as shown in Fig. 21B. The copper ion is ligated by 4-His residues while the zinc ion is ligated by 3-His-1-carboxylate motif.89 The cobalt substitution to zinc didn’t abolish the activity of

Cu/Zn SOD. The crystal structure of the Cu/CoSOD showed that the copper binding site, which is the catalytic metal center, didn’t change much as shown in Fig. 21C. In the proposed reaction mechanism, Cu(II) is reduced to Cu(I) in the first half reaction and regenerated through the second half reaction with two equivalents of superoxide. The substrate binding to the protein is

31 thought the highly conserved Arg141 to replace the water molecules that is shown as red sphere

in Fig. 19 B and C. The NMR study of the reduced Cu/Zn SOD suggested that the two metal

shared His61 is released by Cu(I) and protonated solution. 90 The product inhibition was found in

Cu/ZnSOD. The enzymatic product hydrogen peroxide could oxidize one cysteine and trypto-

phan residues that are close to the active site thus inactivates the enzyme.91

Cu/ZnSOD was found as a bi-functional enzyme. Other than the superoxide dismutation function, Cu/Zn SOD has thiol oxidase activity that coverts the free thiols like cysteine, homocysteine and GSH to their dimer form. 92

Figure 21 The crystal structure of Cu/ZnSOD. A The overall structure B The active site of Cu/ZnSOD C The active site of Cu/CoSOD.

(PDB: 2SOD, 1COB)

1.4.2 Superoxide reductase

Superoxide reductase (SOR) is a small non-heme iron enzyme that only catalyzes the reduction of the superoxide. SOR was originally characterized as a new type of SOD. The activity of SOR is about 1% of the activity of SOD. SOR are employed as superoxide scavenger in anaerobic and microaerophilic bacteria.

The crystal structure of SOR is a homodimer. Two iron centers are presented in each monomer. The ferrous iron that is located in the cupin domain in the active center, which is ligated by 4-His and one cysteine residues which is marked as green sphere in Fig.22A. The ferric

32 ion is marked as orange sphere is a typical rubredoxin iron-sulfur cluster. A calcium ion is presented per homodimer, which his marker as gray sphere. The understanding of the enzyme mechanism has a breakthrough in 2007 when the end-on peroxide intermediate was trapped in

E114A mutation protein crystal. 93 As shown in Fig. 22B, the peroxide is stabilized by order water molecules and the side change of Lys48. The order water molecules are further stabilized by forming hydrogen bonding with Ala45 and Lys48 thus the hydrogen net work is built and stabilized. The hydrogen bonding network protects the product from preceding the O-O bond cleavage by the iron center, which specifies the reaction (Fig.22 B). In the catalyzing reaction, an exogenous protein ligand was proposed to bind with ferric ion in the producing releasing process and the enzyme was regenerated by one electron reduction as shown in the mechanism in Fig.

23. SOR is a different enzyme from SOD but catalyzes a similar reaction. SOR has a more protective active site environment for the hydrogen peroxide to be released than O-O bond cleavage to form iron oxo species. Thus far, iron is the only metal that found active in the enzyme. SOR is stricter with metal selectivity compared to SODs but SOR requires other redox proteins to provide one electron for the reaction. SOR has a highly positive charged active site with one cationic metal with 4-His ligation that allows the enzyme to instantly trap the superoxide to perform the reaction.

Figure 22 The crystal structure of SOR (PDB 2JI3).

Figure 23 The proposed mechanism for SOR. (Adopted from ref 93)

34 1.5 The overview of the dissertation

Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only thiol dioxygenase in mammals. 94 They are the first two enzymes in cysteine degradation pathway. The crystal structure of CDO showed that it is a 3-His non-heme iron cupin enzyme.65 Although no crystal structure for ADO has been reported yet, the sequence alignment of CDO and ADO suggests that

ADO is a CDO homologue that catalyzes the oxidation of cysteamine to hypotaurine. 67 In Chap- ter 3, we will use the spectroscopic tool such as electron paramagnetic resonance (EPR) and

Mössbauer to characterize the iron center properties in ADO. We will also report the crystallization condition for ADO in the same chapter. CDO is better understood compared to ADO; however, no proposed catalytic mechanism for CDO can satisfy all. The argue point of mechanisms is whether or not the iron center needs to form Fe(IV) oxo to insert oxygen to the substrate. Since the substrate of CDO and ADO are both lack visible UV-Vis absorption features, the approval of the mechanism mainly dependents on intermediates trapping by crystallography and Mössbauer spectroscopy both of which are very expensive investment. In Chapter 4, we focus on the mechanistic study of ADO by metal substitution. Copper and nickel doesn’t have the high valent state that is equivalent to Fe(IV) oxo. If the thiol dioxygenase catalytic mechanism requires Fe(IV), then the copper or nickel substituted ADO will be inactive. On the other hand, if Fe(IV) is not required for the catalysis, then copper or nickel substituted ADO should be active. Thus by de- termining the activity of copper or nickel substituted ADO, one can differentiate the two mechanisms. In this chapter, the substrate specificity of human enzymes with copper substitution will also be discussed. In Chapter 5, we will report the current mechanistic understanding of

ACMSD. His228 is localized in the active pocket but is not a metal ligand in ACMSD. We proposed H228 acts as the active site base that deprotonates the metal ligated water for nucleophilic

35 attack on the substrate. The mutants that are generated from site directed mutagenesis of His228 to Tyr and Gly will be characterized by using crystallography and spectroscopy tools.

36 2 MATERIAL AND METHODS

2.1 ADO and CDO

There are two ADO homologues purified. The gene of human ADO was synthesized by a

company and then was cloned to pET 28 plasmid and then transferred into BL21 (DE3) compe-

tent cells. The mouse ADO is a gift from Cornell University Dr XXX’s lab. Human CDO was

purchased from the company. The plasmid was and then transferred into BL21 competent cells

for expression.

2.1.1 The over-expression and purification of ADO

The construction of mouse ADO with His 6-tag and two other expression systems with

different labels have been described elsewhere 67 . In the present work, the E. coli. cells with pre-

viously uncharacterized His 6-tag ADO plasmids were grown in Luria Broth at 37°C with shaking

at 200 rpm in 10 mL and 50 mL size medium containing 100 g/mL ampicillin and 50 g/ml

kanamycin before were transferred into 2 L flasks containing 500 mL medium with the same

concentration of antibiotics. The cultures were grown at 37°C to an OD 600 ~ 0.6. At this point

ADO production was induced by isopropyl-β-thiogalactopyranoside at a final concentration of

0.6 mM. The cultures were grown for overnight at 28°C with shaking. The cells were then har-

vested by centrifugation at 4°C, 8000 × g for 20 min. The cell pallets were dissolved in lysis buffer (20 mM Tris pH 8.5, 300 mM NaCl, 5% glycerol) and the re-suspended mixture was disrupted using the XXX. The cell debris was centrifuged down at 4°C, 30,000 × g, for 30 min and

then for an additional 20 min. The supernatant was injected into an affinity column (Ni-NTA res-

in, 26 × 200 mm). Protein was eluted at a flow rate of 8 ml/min by using a linear imidazole gra-

dient generated from buffer A (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol) and 0–

50% of buffer B (20 mM Tris-HCl pH 8.5, 500 mM imidazole, 300 mM NaCl, 5% glycerol). The

purification was carried out by using an ÅKTA Purifier UPC10 system (GE Healthcare).

37 ADO was eluted out at about 100 mM imidazole concentration. The protein concentrations were

-1 -1 determined spectrophotometrically using the extinction coefficient ε280 = 0.76 (mg/mL) cm

calculated by a web-based protein calculator developed by Christopher Putnam

(http://www.scripps.edu/~cdputnam/protcalc.html ). The anticipated ADO fraction from affinity column was consistent with the theoretical molecular weight of ADO monomer (Mr=28,000) calculated from the amino acid sequence. ADO fraction from the affinity column (Ni-NTA) was concentrated and loaded onto a size exclusion column HiLoad Superdex 75 (26 × 650 mm) and finally stored in 50 mM Tris-HCl, pH 8.5; 25 mM NaCl; 5% glycerol for the assay study. Frac- tions of ADO eluted out from both affinity column and size exclusion column were analyzed for the purity on 12% SDS-PAGE gel.

The oxidized ADO was prepared by oxidizing the as-isolated ADO by 100 mM of potassium ferricyanide for overnight and then desalted the excessive potassium ferricyanide using

3X5nl HiTrap desalting column.

2.1.2 Mössbauer spectroscopy study of ADO.

ADO metal extraction by 1, 10-phanolthroline was unsuccessful. Thus, 57 Fe incorpora-

tion must be achieved during protein synthesis in cell culture. 57 Fe powder was purchased from

Science Engineering & Education Co. The 57 Fe stock solution was prepared by dissolving 57 Fe

57 metal-powder (95.4% of Fe enrichment) in 2.5 M H 2SO 4 under anaerobic condition. The mix-

ture was incubated at 60°C while stirring in an anaerobic chamber until the metal was completely

dissolved in the acid. For preparation of the ADO Mössbauer samples, the metal-depleted Luria

Broth medium was enriched with 57 Fe from the stock solution. A final concentration of 80 M

was used for the cell growth. In this method of preparation all other procedures including the

over expression and purification of ADO were done as described above, except for the last step

wherein the protein was desalted into a 50 mM Tris-HCl pH 8.5, 25 mM NaCl and 5% glycerol

38 buffer using a HiTrap 5 ml desalting column. A 500 L of the concentrated as-isolated protein was transferred into a Mössbauer cup and frozen in liquid nitrogen. Mössbauer spectra were recorded in a weak-field spectrometer equipped with a Janis 8DT variable-temperature cryostat. The zero velocity of the spectra refers to the centroid of a room-temperature spectrum of a metallic iron foil.

2.1.3 EPR spectroscopy of ADO

The EPR samples were prepared in 50 mM Tris-HCl buffer pH 8.5 containing 5% glycerol. Nitric oxide gas was generated by dissolving GSNO power into concentrated hydrochloride solution in a small vial. The released NO gas was added to 1.7 mM of ADO in an EPR tube anaerobically by an anaerobic syringe. After fully interacted with the protein, the EPR tube was frozen in liquid nitrogen. The second sample was prepared by adding 40 mM of cysteamine to

1.7 mM ADO first and then identical amount of the NO gas was added to the ES mixture. After measuring the EPR signal of the second sample, more substrate was added to reach a final 80 mM concentration and this sample was frozen in EPR tube and measured EPR signal. Low- temperature X-band EPR first derivative spectra were recorded in perpendicular mode (TE102) on a Bruker ER200D spectrometer at 100-kHz modulation frequency using a 4116DM resonator.

Sample temperature was maintained with an ITC503S temperature controller, an ESR910 liquid helium cryostat and LLT650/13 liquid helium transfer tube (Oxford Instruments, Concord, MA).

EPR spectral simulations were performed by using a windows-based spin simulation program and a dedicated triplet simulation program, both of which were written by Dr. Andrew

Ozarowski.68

2.1.4 ADO ESI mass spectrometer sample preparation

Ferric ADO was prepared by oxidizing the as-isolated ADO with 100 mM ferricynide for overnight and followed by a desalting step using a 5 ml desalting column. The ESI sample

39 was prepared by incubating 10 mM cysteamine, 200 µM if ferric ADO and 50 mM ethanolamine

which serves as an internal standard in the quantification LS-MS experiment for 1 hour at room

temperature LS-MS samples were prepared similarly as the ESI samples. The enzyme was cen-

trifuged out in both samples after terminating the reaction. Positive model was used in the ESI

experiment and the mobile phase used for the LC-MC MC is 50% of CH 3CN with 0.1% of

HCOOH.

2.1.5 ADO crystallization

Protein for crystallization was purified by four columns: Ni-NTA; size exclusion, Ion ex-

change and then finally polished by the last size exclusion in 10mM Tris pH 8.5. Ni-ADO was

prepared by adding 80 µM nickel chloride to the cells cultured in metal chelated media right be-

fore adding IPTG.

His6-tagged nickel substituted ADO (256 amino acide, ~28 kDa) were screened by about

100 conditions in the crystals screen kits purchased from Hampton reach. 1L of different concentration of protein (5 mg/ml, 7.5 mg/ml, 10 mg/ml, 12 mg/ml, 15mg/ml, 20 mg/ml) was mixed with 1M of mother liquid in the Hang-drop vapour diffusion method. Protein crystals were observed in multiple conditions. Only at conditions of crystals screen number 25 (0.1 M Imidazole pH 6.5, 1.0 M Sodium acetate trihydrate) and 27 (0.2 M Sodium citrate tribasic dihydrate, 0.1 M

HEPES sodium pH 7.5, 20% v/v 2-Propanol) with protein concentration of 15 mg/ml , the protein crystals diffracted well. The fan shape ADO crystals were mounted on the 0.05 mm-0.1mm size of loop. Several cryoprotectants at different concentrations were tested and the crystals were finally frozen with 15% of the glycerol in the mother liquid. Slight varied conditions around the crystal screen 25 (0.1 M Imidazole pH 6.5, 1.0 M Sodium acetate trihydrate) have been used to reproduce the crystals. The crystals that reproduced were soaked with 0.5 M NaI for different time from 1 min – 20 min. The crystal that obtained from 0.2 M Sodium citrate tribasic

40 dihydrate, 0.1 M HEPES sodium pH 7.5, 20% v/v 2-Propanol has not been reproduced so far re-

gardless the varied precipitant or drop size. Further tries are needed. The ADO crystals were re-

produced at the slightly varied conditions of crystal screen number 25. Quick soaking 95 was done

as follows: 0.5 M of NaI was included in the cryoprotectant during the crystal mounting. The

crystals were picked up from the drops and washed with the normal cryoprotectant to get rid of

the protein precipitate. The crystallization of the His6-tagged iron containing protein was also

screen by the screening kits.

The diffraction data of nickel substituted ADO crystals that obtained from crystal number

25 (crystals 1) collected at SER-CAT beam line BM22 at λ=0.93 Å with 180 frames whereas the

crystal that obtained from crystal screen 27 (crystal 2) was and Emory x-ray facility D8 APEXII

at λ=1.54 Å with 360 frames. The two data sets have a resolution of 1.9Å (crystal 1) and 2.2Å

(crystal 2) respectively after the processing with HKL2000. The molecular replacement of ADO was done with CCP4i using nickel substituted CDO structure (PDB 2ATF) and the predicted

ADO PDB model by an online program Phery 2

(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index ). Crystal 1 has been obtained from

the crystal screen number 25 condition is a monomer with a space group of P 21212 1. Crystal 2

which was obtained from crystal screen number 27 is shown as a dimer with a space group of P

121. The figure 1 shows the diffraction map of the crystal 1 and 2. The crystal 1 has a neater dif-

fraction map compared to crystal 2 and the resolution is higher as well.Phenix, CCP4i and coot

are the software that used for the molecular replacement and the attempts of the model building.

2.1.6 Inhibition of the cysteine and homocysteine to ADO

Homocysteine is a substrate homologue for ADO and CDO. Homocysteine was reported

to bind to the iron center in CDO but couldn’t be converted to the oxidized form. Herein we in-

41 cubate homocysteine with ADO in an oxygen electrode and found that homocysteine is inhibitor of ADO

2.1.7 DTNB treatment on ADO

In order to approach to the role of cysteine residues in ADO, DTNB is introduced to the following experiments. DTNB is a cysteine residue modifier which will help to understand the role to cysteine in ADO without any reported crystal structure.

2.2 Crystallization condition of the Fe, Co-ACMSD

Co(II)-H228Y was crystallized following the condition previously established for WT

ACMSD by hanging drop vapor diffusion in VDX plates (Hampton Research). Single crystals suitable for X-ray data collection were obtained from drops assembled with 1 L protein solution layered with 1 L reservoir solution containing 0.1 M Tris-Hcl (pH 8.75), 0.2 M MgCl 2, and

15% PEG 5000. Reservoir solution for Fe(III)-H228Y was modified and contained 0.1 M Tris

(pH 7.0), 0.2 M MgCl 2, and 17% PEG 5000. Crystals were frozen in liquid nitrogen after being dipped into the cryoprotectant solution that contained 30% of glycerol or ethylene glycol in the mother liquid. X-ray diffraction data were collected at SER-CAT beamline 22-ID or 22-BM of the Advanced Photon Source (APS), Argonne National Laboratory, Argonne, IL. Data were collected at 100 K using a beam size matching the dimensions of the largest crystal face. The data were processed with HKL2000. 96 Structure solutions were obtained by molecular replacement using MolRep 97 from the CCP4i program suite 98 with the entire WT Co(II)-ACMSD structure

(PDB entry 2HBX) for Co(II)/Fe(III)-H228Y ACMSD as the search models. Refinement was carried out using REFMAC 99 in the CCP4i program suite 98 for and model-building was carried out in COOT. 100 Restrained refinement was carried out using no distance restraints between the metal center and its ligands. Residue Tyr228 and Gly228 were well-ordered and added to the

42 model based on the 2Fo-Fc and Fo-Fc electron density maps. Refinement was assessed as complete when the Fo-Fc electron density contained only noise.

43 3 SPECTROSPCOPIC, STRUCTURAL ANS MECHANISMTIC CHARATERIZATION

OF THE THIOL DIOXYGENASES

ADO and CDO are essential enzymes in cysteine metabolism. However, both of the enzymes and their substrates are lack the absorption of UV-Vis at visible wavelength thus the approach of the mechanistic study of the two enzymes mostly depends on the spectroscopic tools and crystallization.

3.1 EPR and Mössbauer characterization of cysteamine dioxygenase

Cysteamine dioxygenase (ADO) is a participating enzyme in the synthesis of the essential metabolites hypotaurine and taurine from the substrate cysteamine. Here we describe an optical,

EPR and Mössbauer spectroscopic study of the second discovered thiol dioxygenase in mammals. The majority iron in as-isolated ADO is high-spin Fe(II) with 5/6 coordination enriched in nitrogen and oxygen environment. In this study we found that, the primary substrate of the ADO could reduce the ferric iron into its active ferrous state. The investment of Electron Paramagnet- ic resonance (EPR) and Mass Spectrometry characterization on ADO prove that cysteamine is converted to its dimmer form—cystamine after reducing the ferric to ferrous in ADO. The reduction process is proposed to undergo an Fe(II)-cysteamine radical mechanism. The second interesting finding about ADO is when using NO as a spin probe, the Fe(II)ADO-NO exhibited a mono-nitrosyl (high-spin S = 3/2) iron signal detected by EPR spectroscopy. When cysteamine is added to Fe(II)ADO-NO mixture, a di-nitrosyl iron complexes (DNICs) (low-spin S = 1/2) was formed giving typical DNICs EPR signals. The di-nitrosyl iron complexes (DINCs) formation is rarely reported in non-heme mononuclear iron enzymes.

44 3.1.1 Introduction

Thiols are crucial in regulating oxidative stress, signal translation and transcriptional

processes in cells. Cysteine, cysteamine, GSH and hypotaurine are the widely known sulfur

compounds that function as antioxidants. In thiol metabolism pathways, only two thiol

dioxygenase in mammals have been identified thus far.67 2-Aminoethanethiol (cysteamine)

dioxygenase (ADO) is a non-heme iron dependent enzyme that is responsible for the oxidation of

cysteamine by inserting molecular oxygen to produce hypotaurine (Scheme 1).67-68 Taurine and thiotaurine are known as the side products of the reaction produced by the non-enzymatic oxidation of hypotaurine.69a, b, 101 The existence of hypotaurine and taurine is found in gametes, embryo environment 102 and brain.103 Hypotaurine purportedly was reported to be able to neutralize hydroxyl radicals and prevent sperm lipid peroxidation.104 Taurine is the most abundant amino acids in the brain, which functions as an oxidant scavenger,105 neuromodulation inhibitor,106

regulator of the innate immune system 107 and calcium homeostasis.108 The reaction that generates

hypotaurine from cysteamine by ADO is considered one of the main roots of tautine biosynthe-

sis.109

Upon its first isolation from horse kidney in 1963, ADO was reported to require sulfide,

elemental sulfur, selenium or hydroxylamine to function.69 No further study on this enzyme was

reported until 2007 when Dominy and colleagues described the clone and over expression of

ADO from mouse and rat.67 The recombinant ADO was reported to use ferrous iron as a cofactor

to convert cysteamine into hypotaurine in absence of the inorganic sulfur. Still, information

about the structure and mechanism remain sparse.

ADO is closely related to cysteine dioxygenase (CDO); indeed, both enzymes belong to

the cupin superfamily using 3-His as the metal binding motif.40a, 110 Weston-blot assays of ADO

suggest that it exhibits high expression levels in the brain, heart, kidney, spleen, and other tis-

45 sues.67 CDO, by contrast, is more site-selective: it is expressed at high levels in the liver and much lower levels in the kidney and lung.111 The distribution difference of ADO and CDO ges-

tures toward different physiological roles in biological systems. Specially, CDO catalyzes the

oxidation of cysteine to cysteinesulfinate by inserting two oxygen atoms to the sulfur of cysteine,

and ADO, as stated above, producing hypotaurine and taurine from the substrate cysteamine oxi-

dation. These two enzymes play critical roles in controlling the intracellular thiol level in mam-

mals and generate cysteinesulfinate, hypotaurine and taurine that finally will be converted to the

TCA circle intermediates as shown. ADO and CDO share the same highly conserved metal bind-

ing residues, and both use ferrous iron as a cofactor to function. However, the two thiol

dioxygenase exhibit a low amino acid sequence similarity (14.2-19%) 67 . Notably, CDO is the

better characterized, given a wealth of recent publications on structural, biochemical and model-

ing studies.94, 112 The crystal structure of CDO reveals a four coordination of iron center with

three His residues and a water molecule. An unexpected posttranscriptional cross-link between

two highly conserved residues (Cys93, Tyr157) among eukaryotic CDOs was also reported in the

crystal structure. This cross-link is about 3Å away from the iron center, reportedly enhances the

enzymatic activity of the enzyme by about ten folds, once formed in presence of oxygen 113 . In

2006, a high resolution crystal structure of CDO intermediate bound with its native substrate cys-

teine and molecule of dioxygen was captured in a co-crystallize attempt.114 The hydrophobicity of the enzyme active center highly specifies the substrate and cysteine was found to bidentately bind to the iron center of CDO in the crystal structure. A recent modeling study on CDO’s active center reports that the binding orientation of the sulfur may play a role in driving the enzymatic reaction to go either iron oxygenation or sulfur oxygenation.115 Several catalytic mechanisms based on the crystal structure have been proposed.112e, 116

46 Based upon the recent illuminations of CDO, it is important to understand ADO. In this study,

we characterized the Fe center of mouse ADO by optical X-band EPR and 57 Fe Mössbauer spectroscopy. As-isolated ADO was found, by Mössbauer spectroscopic study, to contain 75% of ferrous iron and 25% ferric iron. Notably, the ferric iron portion of ADO can be reduced to ferrous iron by the primary substrate cysteamine. The EPR study of the iron center by using the spin probe nitric oxide suggests that the binding of the primary substrate mediates the binding sites of the nitric oxide to the iron center forming di-nitrosyl complexes (DNICs) which is the first time detected in mononuclear 3His bounded iron containing protein.

3.1.2 Materials and methods

ADO over-expression and purification The construction of mouse ADO with His 6-tag and two other expression systems with different tags have been described elsewhere. 67 In this work, E. coli. cells with previously uncharacterized His 6-tag ADO plasmids were grown in Luria

Broth at 37°C with shaking at 200 rpm in 10 mL and 50 mL size medium containing 100 g/mL ampicillin and 50 g/ml kanamycin before being transferred into 2 L flasks containing 500 mL medium with the same concentration of antibiotics. The cultures were grown at 37°C to an OD 600

~ 0.6. At this point ADO production was induced by isopropyl-β-thiogalactopyranoside at the final concentration of 0.6 mM. The cultures were grown overnight at 28°C with shaking. The cells were then harvested by centrifugation at 4°C, 8000 × g for 20 min. The cell pallets were dissolved in lysis buffer (20 mM Tris pH 8.5, 300 mM NaCl, 5% glycerol). The re-suspended mixture was disrupted using the cell disrupter. The cell debris was centrifuged down at 4°C,

30,000 × g, for 30 min and then for an additional 20 min. The supernatant was then injected into the affinity column and performed separation (Ni-NTA resin, 26 × 200 mm). 8 ml/min flow rate of the linear imidazole gradient generated from buffer A (20 mM Tris-HCl pH 8.5, 300 mM

NaCl, 5% glycerol) and 0–50% of buffer B (20 mM Tris-HCl pH 8.5, 500 mM imidazole, 300

47 mM NaCl, 5% glycerol) was used in the FPLC program. The purification was conducted by us-

ing an ÅKTA Purifier UPC10 system (GE Healthcare). ADO was eluted out at about 100 mM

imidazole concentration. The protein concentrations were determined spectrophotometrically

-1 -1 using the extinction coefficient ε280 = 0.76 (mg/mL) cm as calculated by the web-based pro-

tein calculator developed by Christopher Putnam

(http://www.scripps.edu/~cdputnam/protcalc.html ). The anticipated ADO fraction from affinity column was consistent with the theoretical molecular weight of ADO monomer (Mr=29,195) calculated from the amino acid sequence. ADO fraction from the affinity column (Ni-NTA) was concentrated and loaded onto a size exclusion column HiLoad Superdex 75 (26 × 650 mm) and finally stored in 50 mM Tris-HCl, pH 8.5; 25 mM NaCl; 5% glycerol for the assay study. Frac- tions of ADO eluted out from both affinity column and size exclusion column were analyzed for purity on 12% SDS-PAGE gel.

Mössbauer Spectroscopy. ADO metal reconstitution was unsuccessful. Thus, 57 Fe incor- poration must be achieved during protein synthesis in cell culture. 57 Fe powder was purchased

from Science Engineering & Education Co. The 57 Fe stock solution was prepared by dissolving

57 57 Fe metal-powder (95.4% of Fe enrichment) in 2.5 M H 2SO 4 under anaerobic condition. The

mixture was incubated at 60°C while stirring in an anaerobic chamber until the metal was com-

pletely dissolved in the acid. Preparation of the ADO Mössbauer samples, entailed enriching the

metal-depleted Luria Broth medium with 57 Fe from the stock solution. A final concentration of

80 M was used for the cell growth. In this method of preparation all other procedures including

the over-expression and purification of ADO were done as described above, except for the last

step wherein the protein was desalted into a 50 mM Tris-HCl pH 8.5, 25 mM NaCl and 5% glyc-

erol buffer using a HiTrap 5 ml desalting column. A 500 L of the concentrated as-isolated pro-

tein was transferred into a Mössbauer cup and frozen in liquid nitrogen. Mössbauer spectra were

48 recorded in a weak-field spectrometer equipped with a Janis 8DT variable-temperature cryostat.

The zero velocity of the spectra refers to the centroid of a room-temperature spectrum of a metallic iron foil.

EPR Spectroscopy. The EPR samples were prepared in 50 mM Tris-HCl buffer pH 8.5 containing 5% glycerol. Nitric oxide gas was generated by dissolving GSNO power into concentrated hydrochloride solution in a small vial. The released NO gas was added to 1.7 mM of ADO in an EPR tube anaerobically by an anaerobic syringe. After fully interacted with the protein, the

EPR tube was frozen in liquid nitrogen. The second sample was prepared by adding 40 mM of cysteamine to 1.7 mM ADO first and then identical amount of the NO gas was added to the ES mixture. After measuring the EPR signal of the second sample, more substrate was added to reach a final 80 mM concentration and this sample was frozen in EPR tube and measured EPR signal. Low-temperature X-band EPR first derivative spectra were recorded in perpendicular mode (TE102) on a Bruker ER200D spectrometer at 100-kHz modulation frequency using a

4116DM resonator. Sample temperature was maintained with an ITC503S temperature controller, an ESR910 liquid helium cryostat and LLT650/13 liquid helium transfer tube (Oxford In- struments, Concord, MA). EPR spectral simulations were performed by using a windows-based spin simulation program and a dedicated triplet simulation program, both of which were written by Dr. Andrew Ozarowski.68

Mass spectrometer sample preparation. Ferric ADO was prepared by oxidizing the as- isolated ADO with 100 mM ferricynide for overnight and followed by a desalting step using a 5 ml desalting column. The ESI sample was prepared by incubating 10 mM cysteamine, 200 µM if ferric ADO and 50 mM ethanolamine which serves as an internal standard in the quantification

LS-MS experiment for 1 hour at room temperatureLS-MS samples were prepared similar to the

ESI samples. The enzyme was centrifuged out in both samples after terminating the reaction.

49 Positive model was used in the ESI experiment and the mobile phase used for the LC-MC MC is

50% of CH 3CN with 0.1% of HCOOH.

3.1.3 Result

The iron content of as-isolated ADO was detected by O-phenanthroline titration. The iron occupancy in as-isolated ADO varies in 35%-62% based on different expression and purification preps.

3.1.3.1 Mössbauer spectroscopy characterization of ADO

To characterize the iron center in the as-isolated his-tagged ADO, the 57 Fe-enriched pro-

tein was measured by 4.2 K Mössbauer spectroscopy. As shown in figure 24A, 23% of the total

iron represented by the red trace is assigned to a ferric doublet ( δ = 0.27 mm/s; EQ = 0.47

mm/s) and 77% of the total are in ferrous state. The 77% ferrous iron is fitted by two quadrupole

II doublets with parameters: Fe A (δA = 1.16 mm/s, EQA = 2.45 mm/s; purple trace in figure 2A)

II and Fe B (δB = 1.26 mm/s, EQB = 2.93 mm/s; green trace in figure 2A). The ratio of the

II II II II Fe A:Fe B is 1:2.2. The δ values of Fe A and Fe B are attributed to high-spin ferrous iron with

penta- or hexa-coordination riching in N/O ligand environment. The Mössbauer parameters also

suggest that the geometry of the iron center is inhomogeneous and the metal ligand environment

is more ionic, which may be due to the binding of one or more labile water liands from solvent

and/or hydroxide ligand(s). To pinpoint the iron center, 50 mM of cysteamine was mixed with

the 2.7 mM 57 Fe-enriched ADO. The Mössbauer spectrum of the substrate-incubated sample is

shown in figure 24B. The ferric species decreased to 16% (Figure 2B, red trace) and 84% of the

iron are in the ferrous state. There are also two ferrous species in this sample.

II The Fe A was still present in the enzyme-substrate mixed sample but the amount de-

creased from 24% of the total iron in as-isolated ADO to 14% in the as-isolated ADO-substrate

II mixed sample. The majority of ferrous Fe B presented in the as-isolated sample (Figure 24A,

50 II green) was converted to Fe C (δC = 1.19 mm/s; EQC = 2.85 mm/s, bule trace in Figure 24C) in

II II the ADO-cysteamine complex. The ratio of Fe A: Fe C is 1:5 after substrate addition to the as-

II isolated ADO. This suggests that Fe B is directly responsible for the substrate binding and form

II Fe C that is representative to ES complex species. Upon comparing the subtracted experimental

spectra, the spectrum of the ES complex (Figure 24C, blue line) shifted leftward. The decrease of

the δ value, from 1.26 mm/s of the enzyme alone to 1.19 mm/s of the ES complex, is in line with

the finding that the sulfur atom of the substrate is covalently bound to the ferrous center. The

reason of the inhomogeneity of the iron center in as-isolated ADO is unknown. However, to

draw comparisons to CDO, the presence of two fractions (with/out a covalent cross-link between

C93 and Y157) in as-isolated CDO has been reported by different groups. The initial iron center

characterization tells us that the iron center in ADO mostly remains at its ferrous state, which

suggests a well folding protein confirmation of ADO protecting the iron center to be oxidized

during multiple purification steps.

3.1.3.2 The reduction of the ferric iron center in ADO by its primary substrate cysteamine

The Mössbauer data clearly shows that substrate incubation reduced the ferric portion in

the sample (Figure 24B). Cysteamine has a reducing thiol, which is presumed to reduce the ferric

iron in ADO to its ferrous state. The reaction is described as follows:

2Fe(III) + cysteamine= 2 Fe(II) + cystamine (equation 1)

To prove the reaction proposed, we prepared a set of ferric ADO EPR samples and meas-

ured them by EPR. As shown in figure 3, the oxidized ADO has a higg spin ( S=5/2) ( g values are

7.0 and 4.3), ferric iron coordinated with protein ligands (A). When mixed the substrate

cysteamine for 20s (Fig. 26B), the EPR signal of the sample is identical to the A. After 5 min

incubation, the high-spin ( S = 5/2) ferric EPR signal of the oxidized ADO with g value at 7.0 and

Figure 24 Mössbauer spectra (4.2 K; 50 mT applied field parallel to γ-radiation) of 57Fe-enriched ADO. A 2.7 mM as-isolated ADO (A); B ADO with addition of 30 mM of cysteamine in the Mössbauer cup; C The overlay of the subtracted spectra of the as prepared (green line) and sub- II II II strate added (blue line) ADO samples. [ Part A: Fe A (purple), Fe B (green), and Part B: Fe A II III (purple), Fe C (blue)] and ferric [Fe (red)]

Table 1 Mössbauer parameters of as -isolated ADO and cysteamine bounded ES complex.

Fe oxidation state δ (mm/s) E (mm/s) % of total iron

as-isolated ADO Fe III 0.27 0.47 23%

II Fe A 1.16 2.45 24%

II Fe B 1.26 2.93 53%

ES complex Fe III 0.27 0.47 14%

II Fe A 1.16 2.45 14%

II Fe C 1.19 2.85 72%

4.3 decreased (Fig. 26C) and completely disappeared after 10 minutes (Fig. 26C). Interestingly,

in spectrum C, a new set of EPR signal with g value at 9.5, 4.9 and 4.3 appeared.This new set of

EPR signal is assigned to a rhombic high-spin Fe 3+ species, the formation of which is due the ligation of the thiol group of the substrate with the ferric iron center in ADO. similar EPR signals were detected previously in Desulforferrodoxin 117 and neelaredoxin 118 . This experiment confirmed that cysteamine is able to reduce the ferric ADO to the EPR silent ferrous state. To further examine the existence of cystamine that is proposed as the oxidation product of the subsrate, mass spectrometry was used to analysis the samples. Electrospray ionization was used for the initial detection of all the ions. Figure 25A is the spectrum of control sample that has substrate and internal standard but without enzyme. Figure 4B is the spectrum of the A incubated with 200

µM ferric ADO. The two samples were prepared as parallel samples. In the positive mode ESI experiment, ethanolamine (m/z 62), cysteamine (m/z 78), cystamine (m/z 153) were all shown in

Figure 25A. The ions at m/z 104 and 122/123 are from buffer (5mM Tris, pH8.0). Some other ions (m/z 93, 108) may due to the fragment of the characterized ions. After the enzyme incubation, the substrate peak (m/z 78) disappeared (Figure 4B), but ethanolamine (m/z 62) adn cystamine (m/z 153) were both detected. A peak with m/z 110 was detected. This 110 ion is assigned to the ferrous enzyme catalytic product hypotaurine. Since both cystamine (m/z 153) were detected in the two spectra, selected ion monitoring LS-MS was used to quantify the amount of cystamine in both samples to see if cystamine was formed after the ferric ADO incubation. Fig- ure 25A shows LC-MS spectrum of the control that was prepared by mixing 10mM cysteamim

53 (m/z = 78) and 50 mM of ethanolamine ( m/z = 62). Three ions were eluted out at 1.29 min (m/z:

153), 1.42 min (m/z: 62) and 1.59 min (m/z: 78), respectively.

C 153 A 100

% 62 78 0 60 80 100 120 140 160

D 62 100 0 1 2 3

% 50 Time (min) 78 153 B 0 60 80 100 120 140 160

E 78 100

62 % 50 153

0 1 2 3 0 Time (min) 60 80 100m/z 120 140 160

Figure 25 The selected ion monitoring LC-MS spectra. A Cysteamine in buffer B The incubation with oxidized ADO with cysteamine. The red line is the internal standard (m/z = 62), the blue presents the substrate cysteamine (m/z = 78) and the olive line presents the cystamine (m/z = 153). C, D and E are the m/z of the mixture at 1.29, 1.42 and 1.59 min

54 The peak area of all the three ions was analyzed. Cystamine content is 4.5% of the internal standard and cysteamine is 37.6% of the internal standard. After the incubation with 200 µM ferric ADO, cystamine takes 11.6% of the internal standard. This experiment confirmed the formation of the cystamine in the equation 1.

Combining the EPR and LC-MS experiment results together, we conclude that cysteamine, the organic substrate of the enzyme, reduces the ferric ion in ferric ADO into its catalytic active ferrous form.

3.1.3.3 EPR spectroscopic study of DNICs formed in ADO

Given that the catalytic active state of ADO is in an EPR-silent ferrous form, we employed nitric oxide (NO) as a spin probe to characterize the enzyme. This method has been used to successfully probe a wide array of ferrous centers in enzymes.

When incubated with NO anaerobically, the ADO-NO complex behaves an axial EPR signal with g values of gy = g z = 4.0 and gx = 2.0 as observed in Figure 5A (the simulation of

ADO-NO complex signal is shown in the dotted line). The signal derives from the well- characterized nitrosyl complex of a non-heme Fe environment bearing N/O ligands, which has a typical high-spin ( S = 3/2) signal with (Fe-NO) 7 electron configuration. In the following experiments, we incubated the ADO-NO sample with 40 equivalent of cysteamine. A new EPR signal

(S = 1/2; g⊥= 2.04, g// = 2.02, g aver. =2.03) appears in place of the original g = 4 high-spin ( S =

3/2) EPR signal (Figure 5B; the dotted line shows the simulation of the observed EPR signal).

The lineshape and the g values of the low-spin Fe-nitrosyl EPR signal is reminiscent of type B of the well-documented dinitrosyl Fe EPR signals 119 found in synthetic and protein-based dinitrosyl iron centers (DNICs) (Table 2). DNICs EPR signals are commonly seen in non-heme iron-sulfur proteins, and are well characterized 120 . For low-spin ( S = 1/2) dinitrosyl iron complex EPR sig-

9 nals, it is formulated as an (Fe-NO 2) unit according to the Enemark and Feltham theorem. The

55 EPR signal at g = 2 ( S = 1/2) of figure 3B DNICs is consider as a high-spin ferrous iron ( SFe = 2)

– coupled to an overall (NO) 2 ligand (S (NO)2 = 3/2) in an antiferromagnetic fashion, resulting in a

120c Stotal = ½. Notably, such a dinitrosyl iron complex was not observed by Pierce et al. in a previous EPR study of CDO.121 Instead, a g = 2 nitrosyl adduct of CDO was assigned to an atypical non-heme low-spin ( S = 1/2) 7 species. The EPR data suggest that the binding stoichiometry of

NO to the Fe center changes according to the presence of cysteamine and this finding adds a new class of the DNICs in biological systems.

Table 2 Summary of EPR g-values of DNICs

Species A-Type B-type

gx, gy, gz g┴, g║ or gav DNIC-HoSF 2.053,2.029,2.011 2.033,2.014 DNIC-L-cysteine 2.04, 2.01 DNIS-BSA 2.05, 2.04, 2.01 DNIS-glutathione 2.045, 2.014 DNIS-homocysteine 2.045, 2.014 DNIS-thiosulphate 2.045, 2.014 DNIS-N-acetylpencillamine 2.045, 2.014 DNIC-Rat liver homogenate 2.03 DNIC-FeFur 2.042, 2.032, 2.017 DNIC-Hb 2.041, 2.014 DNIC-NorA 2.041, 2.018 DNIC-rieske cluster 2.03 DNIC-HiPIP 2.03 DNIC-ADO 2.035, 2.018

A similar prepared sample was characterized by UV-Vis as well (Figure 27). The addition of NO to the as-isolated ADO in an anaerobic corvette generated an absorption band with partial resolved features in the 320 - 370 nm regions that corresponds to the formation of a non- heme

A exp.

sim.

B exp. "/dH χ χ χ χ d

sim.

C exp.

150 200 330 340 350 360 B (mT)

Figure 26 EPR spectra of ADO nitrosyl complex. (A) ADO (600 M) nitrosyl complex in 50 mM Tris-HCl buffer, pH 8.5, in the absence of oxygen; (B) after anaerobic addition of cysteamine; (C), the solution as in B, after further addition of excessive NO. Instrument conditions: temperature 10 K, microwave frequency 9.64 GHz, microwave power 1 mW, modulation frequency 100 kHz, modulation 5 G. Arrows indicate g = 4.3. Simulations of the spectra (dotted lines) are plotted under the experimental data.

3 c

Absorbance b 1 a

0 300 400 500 Wavelength (nm)

Figure 27 The UV-Vis absorbance of ADO (a); ADO-NO (b) and ADO-NO-S (c)

57 Fe-nitrosyl complex. Further addition of NO did not significantly change the absorption

(data not shown). However, further addition of cysteamine resulted in a blue shift concomitant

with a significant increase in the absorbance of the Fe-nitrosyl complex. The shift of λmax to a high-energy region is consistent with substrate binding to the Fe-nitrosyl complex.

3.1.4 Discussion

Herein we characterized and reported the biochemical behaviors of ADO using spectroscopic tools for the first time. We have noticed that even though ADO and CDO are always being talked together as “sister enzymes” that are the only know thiol dioxygenases in mammals, they behave quite differently in terms of the biochemical features in vitra . As mentioned in the

introduction part, CDO has a cross-link between C93 and Y157 in its crystal structure, which

yields two bands of CDO on SDS-PAGE. The formation of C93-Y157 cross-link is not reducible

by DTT. The cross-link formation in CDO is posttranscriptional and could be generated in vitro by incubating the non-crosslink protein with exogenous iron and cysteine. The C93-Y157 crosslink increases the enzymatic activity by ca. 10 folds. Although structure of ADO has not been reported, our SDS-PAGE result of purified ADO didn’t show two protein bands next to each as shown in CDO, thus ADO either doesn’t have the same type of covalent bond or the crosslink doesn’t affect the protein mobility on SDS-PAGE, instead, ADO exhibits different confor- mations in native gel and could be altered by addition of reducing agent.

The mössbauer spectrum of as-isolated ADO suggests that the ferrous iron exhibits a penta- or hexa-coordination, which means that more than one water molecules are ligated to the iron other than the three histidines. This type of active site geometry makes the oxygen binding more difficult than in CDO when the organic substrate is absent, which may explain why ADO is more resistant to Fe center auto-oxidation but instead its protein residues such as Cys residues.

58 Although no ADO structure has been reported yet, one still can conclude that the overall structure of ADO is very protective to the ferrous center from the oxidizing agent.

3.1.4.1 Ferric ion reduction by the substrate

The EPR and mass spectrometry experiments prove that the substrate if ADO is able to reduce the ferric iron to its catalytic active ferrous state. The new EPR signals with g values of

9.7, 4.88 and 4.3 in Figure 3C suggests that the reduction is performed by direct ligation between substrate and the ferric iron.

Similar EPR signals have been detected in ferric CDO with the incubation of cysteine but no reduction reaction occurred 122 . The reason why cysteine cannot reduce the ferric CDO is not clear. Since cysteine and cysteamine have similar reducing potentials, the different observations for ADO and CDO may actually come from the structure difference. Cysteine binds to the iron center bidentately through the nitrogen and the sulfur. The nitrogen in cysteine is about 3.08 Å away from the sulfur in PDB structure (3ELN). The two oxygen atoms in the carboxyl group are hydrogen bounded to the Arg60 that stabilizes the substrate during reaction. In cysteamine the carboxyl group is missing compared to cysteine. The nitrogen is 4.08 Å away from the sulfur in cysteamine after the energy minimization. This may suggests that cysteamine may not bind to the iron in the same bidentate manner of cysteine binds to CDO; instead it binds to the iron center through the sulfur (Figure 3C) and only sulfur.

The substrate reduction phenomenon was also seen in tryptophan 2,3-dioxygenase (TDO) in which the substrate is not ligated to the iron center.123

3.1.4.2 Interaction with NO and substrate binding manner proposal in ADO

Nitric oxide has very wide physiological effects in body and alters many signaling pathways through chemical modifications, such as the addition of S-nitrosothiols and nitrosotyrosine to target proteins altering various biological pathways.124 Ferrous iron can form complexes with

59 different forms of NO, e.g. NO +(S=0), NO · (S=1/2), NO - (S=0), NO - (S=1), NO 2- (S=1/2) in solution. The iron d orbital electron plus the π* electron of NO can give a total d orbital electron of

7 9 6-8. (Fe-NO) and (Fe-NO 2) are EPR visible species and both have been detected and characterized in the mononuclear non-heme iron protein 125 . DNICs acts as a NO donor have been selected as a basic material for pilot-scale production of a hypertensive drug that is recommended for the second phase of clinical trials. 126

The DNICs are well studied in NO storage proteins such as iron-sulfur proteins with

125b Fe(cys) 2(NO) 2 ligation . There are mainly 4 types of DNICs reported in literature but the

mononuclear DNICs are major species responsible for the biological effects. 127 Thus far, the

characterization of the mononuclear DNICs is base on synthesized compounds. 128 The clinical

study of the DNICs is generated by mixing ferrous iron with cysteine or glutathione in aqueous

solution, which is not stable to achieve the real function of mononuclear DNICs. 126 Although

peptide-bound DNICs have been investigated, the DINCs formed in ADO is the first protein

based mononuclear DNICs 129 . It may not directly relate to the cysteamine oxidation catalytic

mechanism but the DNICs formation in ADO opens a door of correlating ADO with NO related

physiological study. The unique phenomenon in ADO DNICs is the formation involves in the

binding of its primary substrate cysteamine.

ADO is due to the extrinsic thiol binding of cysteamine, and presumably deprotonation

9 of cysteamine is required to form (Fe-NO 2) species with ferrous iron and two NO molecules.

The reason that CDO doesn’t form DNICs is that the valent of ferrous iron is saturated by three

histidines, bidentate ligation of the cysteine and one NO molecule. The DNICs formation in

ADO also suggests that cysteamine is monodentately bounded to the iron.

60 3.2 The structure characterization of the ADO

Thus far, no structure of ADO has been reported. The view and understanding of the

structure of an enzyme is very important to approach the mechanistic study and the regulation

roles in vivo thus we want to pursue the structure of ADO while doing the biochemical charac-

terization .

3.2.1 Crystallization of a de novo crystal structure of a mononuclear Non-heme Iron

Cysteamine dioxygenase (2-aminoethanethiol (ADO)) is the second thiol dioxygenase in mammals that so far reported. ADO is a ~28 kDa non-heme mononuclear iron dependent enzyme that catalyzes the oxidation of cysteamine in presence of molecular oxygen to generate hypotaurine. There are two constructions for the recombinant mouse ADO used in the crystallographic study, one with His6-tag at the N-terminal while the other contains a Xa-factor cleavage site after His6-tag. Both of the two constructions are transferred and over expressed in E.coli .

Nickel was used to substitute iron in both proteins. Two His6-tagged ADO crystals with nickel substitution diffracted to 1.9 Å and 2.4 Å resolutions respectively after processed with

HKL2000. The molecular replacement was tried with CDO (PDB 2ATF) that has about 19% identity with ADO and the built model of ADO by an online software phyre2; however, neither of the two model is suitable to solve the phasing.

Native iron containing ADO was used for screening set up by robotic system. The initial condition that has 0.1 M Hepes, 0.2 M NaCl, and 20% PEG 3500 gave crystal needles.

Further variation with 0.1 M cacodalate, 0.2 M NaCl, and 20% PEG 3000 generated plate form crystals that diffracted at 2.2 Å.

The blast results showed that CDO is the closed structural related protein thus the

HKL2000 processed data sets were used further pursuing the structure of ADO. Unfortunately, the sequence identity of two proteins is about 19%, which at least 30% of sequence identity is

61 normally required at for molecular replacement. The R work and R free were initialed from 58%

and 60%, which suggests that model is most likely wrong. We then used phyre2 to search the

model of ADO based on the sequence. The built model by the online software is also used as the

model for the molecular replacement with CCP4i. Phenix but none of those structure methods

have given an acceptable R work and R free to overcome the phasing problem.

There are four ways to solve the phasing for a de novel , which are Se-Met substitution, heavy metals soaking, signal wavelength anonymous signal for transition metal containing proteins and sulfur phasing. The most popular one is growing the protein with M9 media that is substituted with Set-Met. The Se-Met will replace the Met and provide selenium to help solve the phasing. The heavy metal soaking refers to incubating the crystals with Br -, I- , Pt, Au, Hg, etc. for short time. The third method is sometimes used for the metal containing proteins. The sulfur phasing is using sulfur atom anonymous signal from cysteine or methionione in the protein. Among the four methods, sulfur phasing requires very high quality crystals with good diffraction data while the signal wavelength metal signal requires the metal to protein ratio is high enough to be observed. We have tried the Se-Met substitution and heavy soaking with Br -, I-, Pt,

Au, and Hg. Very disappointingly, Se-Met substituted ADO doesn’t crystallized at the none of the original or the varied conditions while the heavy metal soaking either crushes the crystals or fails to bind with protein. Further attempts are still undergoing.

3.2.2 Characterization of the role of cysteine in ADO

The expression system of ADO was constructed by Dr. Stipanuk’s group. Three versions of ADO were constructed. We chose to work with the His 6-tagged construction since other two versions contain large tag for stability. When purified by size exclusion column, ADO shows multiple peaks on the elution profile (Fig.28). The SDS-PAGE result shows that the first two fractions have two major bands while the third fraction only shows one band. When DTT is add-

62 ed into the sample loading buffer, all three fractions only show one single band at equal position on the SDS-PAGE. This suggests that, the three fractions of ADO eluted from the size exclusion column are different multimer forms of the protein. It also suggests that the polymerization is a redox-dependent reversible process. To further elucidate the role of the DTT in the conformation change of ADO, 20 mM of DTT was used to pre-treat the as-isolated ADO after the purified by affinity column, then, 5mM of DTT was included into the elution buffer during the size exclusion purifying procedure. As shown in Figure 28, in the presence of 5 mM DTT, the first two fractions shifted to the position of the third fraction suggesting that DTT is able to maintain the protein in the uniform conformation. Native gel experiment was also invested on this protein.

Two major fractions presented by P1 and P2 and one native marker sample were loaded on the native page.

Another interesting phenomena of ADO is when incubated with DTT, the enzyme turned into blue color and lost activity. The sequence ligament of the ADO with CDO shows that the second sphere Tyr157 in the active site of CDO is conserved in ADO. The function of the Tyr residue was proposed to stabilize the oxygen molecule through hydrogen bond. The mutations of the Tyr 157 in CDO resulted in inactive enzyme. The blue color in protein is an indication of the dopa formation by oxidizing Tyr residues. In ADO amino acid sequence, there are 8 Tyr residues thus we propose the DTT treatment oxidized one or some of the residue(s) to dopa. Our NBT staining result of the ADO before and after DTT treatment confirmed there indeed is dopa formed in ADO after DTT treatment, which resulted in inactive form of enzyme.

) w ithout DTT w ith DTT 280 nm 280 A ( bsorbance bsorbance A

100 150 200 250 Elution volume (ml)

Figure 28 Size exclusion purification profile of as-isolated ADO with and without DTT in the elusion buffer and the SDS results.

The gel filtration separation and the SDS-PAGE results suggest that the cysteine residues that locate on the surface of ADO may be involved in the above phenomenon and enzymatic activity. In this study, DTNB was used as the cysteine modifier. After the incubation of the 10 molar equivalent of DTNB, the as-isolated ADO was then run through the size exclusion column.

As shown in Fig.29, without DTNB the protein are mainly separated for four fractions.

The DTNB modification changed the separation profile by dragging the middle fraction into two side fractions suggesting that the as-isolated ADO is a polymerization mixture part of which is caused by the cysteine residues. The activity assay showed that all the fractions eluted from the size exclusion column with or without DTNB treatment are active (table2). The second fraction with DTNB treatment has the highest activity where the first fraction has the lowest activity.

200

ADO+DTNB ADO 150

100

mAU 280

A 50

50 100 150 200 250 Elution Volume (ml)

Figure 29 The gel filtration profile of ADO with/out DTNB incubation.

Table 3 The activity of ADO fractions eluted from size exclusion column. Protein rate (nmol/mg/min) ADOP1 1208.2 ADOP2 2382.1 ADOP3 1636.2 ADOP4 1432.3 DTNBP1 930.3 DTNBP2 2812.8 DTNBP3 1496.8

In order to find out the structural reason for the activity difference, CD spectra of the different fractions were pursued. The CD spectra of the four fractions of as-isolated ADO are very similar with similar intensity. When compare the spectra of each fraction with/out DTNB treatment, it shows in Fig.30 that the second fraction with DNTB treatment has the highest intensity suggesting this fraction is the best folded, which further suggests the folding of the protein is somehow related to cysteine residues and are very important for the enzymatic activity. The

ADO crystal structure is still not reported thus the location of the cysteine is unknown. To sum- marize all the phenomena observed in this topic, we draw a picture as shown in Fig31 to propose

65 the structural role of cysteine in ADO . The dimer form is the most active formation, which may

be bridged by disulfide bonds. ) -1

) 20 A ADOP1 -1 B 40 ADOP2 0 residue -1

ADOP3 residue 20 ADOP4 -1 -20 dmol 2 dmol 2

0 -40

(degcm DTNBP1

-3 -3 -60 -20 (degcm DTNBP2 -3 -3

x10 DTNBP3 -80 x10 [θ] -40

200 210 220 230 240 250 [θ] 200 210 220 230 240 250 Wavelength (nm) ) Wavelength (nm) -1

40 )

-1 40 C ADOP1 D ADOP2

residue 20 DTNBP1 20

-1 DTNBP2 residue -1 0 dmol

2 0 dmol 2 -20 -20 (degcm -40 -3 -3 (degcm -3 -3 x10 -40 -60

200 210 220 230 240 250 x10 [θ]

Wavelength (nm) [θ] ) 10 -1 -80 E ADOP3 200 210 220 230 240 250 DTNBP3

0 Wavelength (nm) residue -1 -10 dmol 2

-20 (degcm -3 -3 -30 x10

[θ] 200 210 220 230 240 250 Wavelength (nm)

Figure 30 The CD spectra of ADO. A and B are the CD spectra of the four fractions of non-modified and DTNB modified ADO, respectively; C, D and E are the comparison of the each fraction with or without DTNB modification .

66 SH SH SH

SH SH SH SH HS HS SH HS HS HS HS HS HS Not very active

SH SH SH

SH DTNB HS DTNB DTNB DTNB HS HS HS DTT DTNB HS DTNB HS SH Very active SHDopa DTNB DTNB SH DTNB HS Inacive Very active HS

HS SH SH

SH HS Active

Figure 31 The summary of the role of cysteine residues in ADO.

The as-isolated ADO is mixture of all polymerization mixture. The large complex in as- isolated ADO is caused by unspecific disulfide bond and hydrophobic interactions between dimer form. The large complex could be unpacked by DTT or DTNB. DTT treatment not only dis- sociates the huge complex, breaking the disulfide bonds it also modifies the Tyr residues in ADO causing dead form enzyme. The huge complex, dimer and monomer are in equilibrium understand buffer condition.

67 4 THE INVESTIGATION OF METAL AND SUBSTRATE SPECIFICITY OF THIOL

DIOXYGENASE

Metalloenzymes are versatile catalysis. Some enzymes are much conserved with metal

while others can use different metals to do the same reaction or different reactions. CDO has

been reported as an iron dependent dioxygenase. In this study, the metal and substrate specificity

will be invested in ADO and CDO.

4.1 ADO is not a iron dependent non-heme enzyme

ADO was reported as a non-heme iron dependent dioxygenase with 3-His metal binding

motif. However, the 3-His metal binding motif is not an iron conserved motif. The metal substi-

tution followed with activity assay of ADO showed that ADO is not a strict iron dependent en-

zyme. The activity of apo ADO could be rescued by Cu(II) and Ni(II) reconstitution at different

levels. The kinetic parameters were obtained from the study of the steady-state by oxygen elec-

trode and the product formation was confirmed by mass spectrometry.

4.1.1.1 Introduction

Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are known non-

heme iron dependent dioxygenase in cupin superfamily. ADO is encoded by the gene Gm237,

which is identified as a hypothetical murine protein homolog of CDO from the DUF1637 protein

family. It was first cloned and expressed by Dominy et al. in 2007.130 Although ADO and CDO are homolog, they only share about 14% amino acids similarity and they don’t share substrates.

ADO itself and its substrate both are lack of the UV-visible absorption like the CDO case so the characterization of the protein is more difficulty to approach.

The metal binding motif in metalloenzymes varies depending on the enzymatic functions.

The most popular c metal binding motif is 2-His-1-carboxyl motif that are indentified in ring

68 cleavage dioxygenases,131 α-KG-dependent taurine dioxygenase 30, 132 and Rieske

dioxygenases.133 3-his-1-carboyxylate, which is the second popular metal binding motif, is most-

ly employed by cupin superfamily members.8a, 50a Enzymes like quercetin 2,3-dioxygenase

(QDO) 134 and acireductone dioxygenase (ARD) 51 (PDB entry 1VR30) both utilize this type of

metal binding motif to function. The first crystal structure of CDO revealed a 3-His metal bind-

ing motif, which immediately draw the attention of the field.135 The sequence blast search

showed a sister enzyme of CDO, which is found to have cysteamine dioxygenase activity named

ADO has the same metal binding motif and catalyzes a similar thiol dioxygenation reaction. The

structure of ADO is not yet reported. However, two ring cleavage enzymes named diketone

cleaving dioxygenase ( Dke1; PDB entry: 3BAL) and gentisate 1,2-dioxygenase (PDB entry:

2D40) were both reported as 3-His monoiron dependent enzymes.58

The dioxygenases with 2-His-1-carboxyl and 3-His-1-carboyxylate motif are very well

characterized. The third type of metal binding motif that only has 3-His derives from the 3-his-1-

carboyxylate binding motif by breaking the conservation of the carboxyl in the cupin motif.40a

It’s common that one enzyme can use more than one type of the metals to function.

Homoprotocatechuate 2,3-dioxygenases form Brevibacterium fuscum (Fe-HPCD)

and Arthrobacter globiformis (MndD), are homologues with different native metals ions. The

metal swapping of the two enzymes results in fully active enzymes with similar kinetic parame-

ters as the as-isolated form.16 Similarly, the 3-his-1-carboyxylate containing QDO isolated from

Aspergillus is reported to bind Cu(II) 134 to function but the homologue enzyme from Bacillus

subtilis binds Fe(II) that could be substituted with Mn(II), Fe(III), Co(II), Ni(II) and Cu(II) with comparable activity with the native form.47a, 49

The 3-His iron binding dioxygenase named Gentisate 1, 2-dioxygenase (GDO) and CDO are known as iron dependent enzymes. GDO catalyzes the oxidation of several substrates but on-

69 ly iron is active in the protein. CDO is able to bind nickel shown in the first crystal structure;

however no activity was observed with other metals in CDO. The third crystallized 3-His con-

taining dioxygenase, Dke1 dioxygenase, is a promiscuous enzyme. The native Fe(II) enzyme has

two functions, the first function is converting 2,4-pentanedione and O 2 into methyglyoxal and

acetate and the second function is hydrolyzing 4-nitrophenylesters of short-chain alkanoic acids.

The metal substitution study showed that Zn(II) enzyme only has the hydrolyzing activity but is

ten times higher than native enzyme.70

Due to the lack of UV-visible absorption of the substrate and the product of CDO, no kinetic intermediate has been trapped in solution; however, a persulfenate intermediate was trapped in single crystalline.114 The crystal structure of the intermediate shows that the iron center is bidentate legated by the cysteine and the iron center forms three member ring with the sulfur from cysteine and the proximal O from dioxygen. The mechanism proposed based on the crystalline intermediate differs from other mechanism proposed for other non-heme dioxygenase that, neither ferryl-oxo nor the O-O bond cleavage is required in the turnover.114, 136 (update with the recently publication on CDO). However, some research groups proposed a different catalytic mechanism for CDO that requires the iron center to form Fe(IV) species to insert oxygen to the substrate.

In chapter, we used metal substitution approach to differentiate the two mechanisms for thiol dioxygenase. The transition divalent metal ions, which don’t have the IV oxidation station, will be substituted to apo ADO. We found that Cu(II)- and Ni(II)-substituted ADO has detectable activity thus the catalytic mechanism of thiol dioxygenase very unlikely go through the

Fe(IV) state during catalysis. Moreover, the copper reconstituted ADO gained the catalytic activity toward cysteine and homocysteine. Similar behavior was found to CDO as well, the differ-

70 ence is after copper reconstitution CDO lost cysteine oxidation catalytic activity but gained new activity on cysteine homologues.

4.2 Materials and Methods

4.2.1.1 Protein construction and expression

ADO was purified as shown elsewhere.130 CDO that used in this study is a homo sapiens protein

(hCDO). The constructed cell glycerol stock was purchased from Arizon State University

(http://dnasu.asu.edu/DNASU/GetCloneDetail.do?cloneid=84609&species= ). The expression of hCDO started from the single colony from the plate. 100 ug/mL ampicillin was included in the media culture stage. The protein was induced by 0.5 mM IPTG at 28°C for overnight when the

OD 600 reached 0.6. The harvested cells were then disrupted in presence of the 2 mM PMSF. The cell lysate was then centrifuged at 27,000g for 40 min. The supernatant was then loaded onto Ni-

NTA (26X40) pre- equilibrium column. A liner gradient program was run with buffer A (20 mM

Tris-HCl pH8.0, 0.2 M NaCl and 5% glycerol) and buffer B (20 mM Tris-HCl pH8.0, 0.2 M

NaC, 0.5 M imidazole and 5% glycerol). The hCDO fraction is collected from and concentrated before loaded onto a HiLoad Superdex column (26X65).

4.2.1.2 Protein reconstitution

The as-isolated protein was first treated with o-phananthroline and EDTA, respectively. Desalt- ing was done using 3X5 mil Hitrap desalting columns purchased from GE healthcare.

The metal reconstitution was preceded by incubating the divalent metal salt stock solution with the apo protein at 4°C for overnight. The precipitation was centrifuged at 13,000 rpm for 2 min.

After desalted the extra metal salt by desalting column, 0.1 mM EDTA was added and desalted out to strip out the unspecific bounded metal ions.

71 4.2.1.3 Activity assay

The activity of ADO and hCDO was detected by monitoring the oxygen consumption by Oxygen

Electrode. The instrument was first calibrated at room temperature. The reaction buffer for ADO is 50 mM Tris-borate buffer pH 8.5 with 50 mM NaCl.

4.2.1.4 ICP-OES

4 mL of 10 µM protein was prepared in 10 mM Tris-HCl buffer pH 8.0 using ultra purified ICP water. 5, 50 and 500 ppb standard solutions with Fe 2+ , Cu 2+ , Mn 2+ and Ni 2+ metal ions were prepared and run first. After the liner stand curves were obtained, the protein samples were injected into the machine.

4.2.1.5 ESI-MS

The samples were prepared by incubating ADO or hCDO with 50 mM of cysteine and homocysteine at room temperature with oxygen bubbling for 20 min.

4.2.2 Results

The as-isolated ADO was purified as described previously. Apo ADO was obtained by treating as-isolated with EDTA and 1, 10-phanonthroline. The reconstitution of the several divalent metals was performed on apo ADO. The metal content of samples of apo ADO reconstituted with Fe(II), Cu(II), Mn(II), Ni(II) and Co(II) were analyzed by ICP-OES experiment (Table 3). It is shown that the reconstitution with Co(II) and Mn(II) can only recover less than 10% per protein, suggesting these two metals are not favorable by the 3-His metal binding motif in ADO.

Not surprisingly, the reconstitution of Fe(II) and Ni(II) recovered fully or most of the metal content. Cu(II) is a histidine rich metal motif favorable metal, thus the reconstitution with Cu(II) recovered half of the metal in ADO.

72 Table 4 Metal contents of reconstituted ADO.

Protein Fe Cu Ni Mn Co As-isolated ADO 0.44 0.04 0.35 0.03 0.01 Apo ADO 0 0 0 N/A N/A Fe(II)-reconstituted ADO 1.05 0.02 0.02 0.01 0.01 Cu(II)-reconstituted ADO 0.01 0.55 0 N/A 0 Ni(II)-reconstituted ADO N/A N/A 0.88 N/A N/A Mn(II)-reconstituted ADO N/A 0 0 0.08 0 Co(II)-reconstituted ADO N/A N/A N/A N/A 0.03

Table 5 The apparent parameters of ADO with different metal constant.

Protein app app (min -1) app app -1 -1 3 Km (mM) kcat K m k cat (s M )·10 Apo ADO N/A N/A N/A Fe(II)-reconstituted ADO 0.556 ± 0.094 34.3 ±1.80 1.03±0.32 Cu(II)-reconstituted ADO 0.410 ± 0.092 78.6 ± 2.8 3.2 ± 0.10 Ni(II)-reconstituted ADO 0.160 ± 0.007 9.4 ± 0.11 0.98 ± 0.26

B A 45 30 30

20 V/[E]

V/[E] 15 10

0 0 0 1 2 3 4 5 Cystamine concentration (mM) 0 1 2 3 4 5 Cysteamine concentration (mM)

10 C

6 V/[E]

0 1 2 3 4 5 Cysteamime concentration (mM)

Figure 32 Micaleas-Menten curve of metal reconstituted ADO.

73 The activity of different metal reconstituted ADO was then determined by oxygen electrode

and the kinetic parameters are listed and compared in Table 5. Apo ADO is inactive. The Km

value of Fe(II) and Cu(II) reconstituted ADO are similar but Ni(II)-ADO has two folds low-

er Km. Interestingly, Cu(II)-ADO has higher kcat value with 3 folds higher catalytic efficien-

2+ cy than native metal. It is known that Cu catalyzes the oxidation of free thiols to form disulfide bond. In the control experiment, oxygen consumption was also observed when cysteamine or cysteine was mixed with Cu 2+ . Since the oxygen electrode monitors the oxygen consumption, which is an indirect way to examine the reaction, it’s not clear if it is Cu 2+ itself or CuADO are doing the catalysis to cysteamine. Thus, we determined the production formation of Cu(II)-ADO and Cu 2+ mixed with cysteamine by ESI mass spectrometry, respectively. To be comparable, Cu(II)-ADO or Cu 2+ alone was prepared in 5 mM Tris buffer, which was then mixed with 60 mM cysteamine for 30min. The ESI spectra were shown in Fig. 33 and 34. The main Tris peak is at 122. The 104 peak is the dehydrolysis fragment of Tris, whereas the 144 peak is Tris bonded with sodium cation. The substrate cysteamine is at 78 and 61 is the deamination fragment of cysteamine. The 108 peak is the fragment of the ADO enzymatic product hypotaurine and 153 peak is the dimer form of cysteamine. Us- ing the 122 Tris peak as the internal standard, the relative quantization of reaction product could be represented by the ratio of the 108 peak to 122 peak. The two samples have similar products which are hypotaurine and the disulfide cystamine but CuADO has much higher product formation than Cu 2+ .

Although the ESI experiment confirmed the hypotaurine is product of CuADO, it also con-

firmed that Cu 2+ alone in buffer does the same catalysis thus we still don’t know if the activ-

ity of the CuADO is due to Cu 2+ in buffer or due to the active site bounded form. To distin-

guish the two, we applied histidine modifying reagent Diethylpyrocarbonate (DEPC) to our

74 experiment. As shown in Figure 35, the modification position on histidine is the same as the metal binding site. original solution QtofMicro 06-Sep-201316:04:52 YAN_BUFFER+S+CUADO_AILIU_09062013_ESI-POS 78 (1.449) Cm (44:85) TOF MS ES+ 108.00 100 2.73e4

153.07

122.09 %

77.02 136.04 60.99 104.08 110.00 78.03 155.07 68.50 123.10 0 m/z 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160

Figure 33 The ESI spectrum of CuADO with cysteamine mixture after 30 min incubation. original solution QtofMicro 06-Sep-201316:08:39 YAN_BUFFER+S+CU_AILIU_09062013_ESI-POS 96 (1.788) Cm (83:147) TOF MS ES+ 122.09 100 3.51e4

108.00 % 153.07

60.99 78.03 104.08 77.02 110.00 123.10 136.04 155.07 144.08 0 m/z 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160

Figure 34 The ESI spectrum of Cu 2+ with cysteamine mixture after 30 min incubation.

Thus, if we modify the apo protein with DEPC, we should detect little activity if Cu 2+ binds to the active site. If we modify the protein after the metal reconstitution, then the activity will not be affected by DEPC as shown in Fig. 36.

Figure 35 The DEPC modification reaction on histidine. His His N His His His N His N H N N N His His N His His HN N N NH N

DEPC-His His-DEPC N DEPC-His N His-DEPC DEPC-His His-DEPC N N N N N DEPC His-DEPC DEPC-His N N His-DEPC DEPC-His N N N DEPC DEPC

Fe/Cu reconstitution

Active Inactive

Figure 36 The experiment design of using DEPC histidine modifying reagent.

Wash 2+ Wash Apo ADO + 80 e.q. of DEPC Recustitute with 2 e.q. of Cu Activity Assay

2+ Wash Wash Apo ADO + 2 e.q. of Cu CuADO + 80 e.q. of DEPC Activity Assay

Figure 37 The illustration of DEPC modification experiment.

As demonstrated in Figure 37, apo ADO was modified by DEPC before and after the metal reconstitution. The activity of the two was then measured by oxygen electrode with three repeats. The activity assay results of CuADO and FeADO are compared in Fig. 38. The DEPC modification does not affect the activity if the active site is pre-occupied by metal ions. Howev- er, the enzyme activity dropped 90% if apo protein was modified by DEPC before metal reconstitution. These experimental results confirmed copper binding to the active site is required for the enzyme activity.

1.0 1.0

0.8 0.8

0.6 0.6

Activity % 0.4 Activity % 0.4 0.2 0.2 0.0 0.0 FeADO Apo ADO Apo ADO CuADO Apo ADO Apo ADO Fe DEPC Cu DEPC DEPC Fe DEPC Cu

Figure 38 The activity of Fe and CuADO after DEPC treatment.

4.2.3 Discussion

Metalloenzymes have developed diverse metal binding motifs to function. Some of the enzymes have both metal and substrate specificity, which are mostly found in heme proteins. The non-heme iron enzymes instead, are found more tolerant to the metal core diversity. The enzyme function is determined by four main structural factors: 1) the metal core; 2) the metal binding motifs; 3) the second sphere residues and 4) the electron statistics. The mechanisms of the non- heme iron dependent dioxygenases with different metal binding motif have been widely studied.

There are several distinguish mechanistic studies on the enzymes mentioned above 50a . The understanding of enzymatic functions lays in enzyme structure features and the substrate biochemical features. The mechanism of the non-heme iron dependent dioxygenases could be summarized as the understanding of the four major puzzles: 1) the oxygen activation; 2) the role of the metal ion; 3) the O-O cleavage manner and 4) the function of the second sphere residues.

In ADO, we found that Cu(II) is active, even more active than the native metal ferrous iron. In the reported literature, no high-valent copper species has been seen in biological system.

78 Cu(II)- and Ni(II)-substituted ADO have detectable activity, thus the iron center in ADO very unlikely goes through high valent Fe(IV) state during catalysis. Dioxygenases catalyze the breaking of C-C or C-H bond by inserting both or just one oxygen into the primary substrate. In al- most all of the mechanisms that have been proposed, the O-O bond is cleaved either through the high valent metal species formation or the pulling force from metal and substrate. Different from other examples, CDO and ADO oxidizes a small polar compound on the sulfur atom that is electron rich thus it is more likely the two oxygen atoms are spontaneously inserted into the sulfur group on the substrate. 137

4.3 Initial characterization of human enzymes

CDO is a non-heme iron dependent dioxygenase that inserts molecular oxygen into the primary substrate cysteine in presence of oxygen. ADO is mostly expressed in brain and heart and other various organs in mouse, differently, CDO is only intensively expressed in liver.135

ADO with cysteine dioxygenase (CDO) was the only reported thiol dioxygenases in mammals.130 CDO is the first enzyme in the cysteine metabolism pathway. The crystal structure of as-isolated CDO reveals a ferrous iron that is ligated by three histidine ligands and one water molecule in tetrahedral geometry.135 This is the first reported 3-His metal binding motif in non heme iron proteins. Another unique structural feature in CDO is that a cross link between C93 and Y157 is formed through covalent bond. When the recombinant CDO was purified by the affinity column, the SDS-PAGE shows two bands with slightly different molecular weight. This mystery was uncovered after the crystal structure was solved. The fact is that in the aerobic condition, part of the protein forms the cross-link between Cys93 and Tyr157. In anaerobic condition, there is only one band on the SDS-PAGE that has the molecular weight corresponding to non-crosslink protein. In as-isolated CDO, it presents a mixture with 7:3 ratio of non-crosslink form vs. crosslink form. With the crosslink, CDO was found 10 folds more active.138 The se-

79 quence alignment of ADO and CDO from different sources suggests that the 3-His iron binding

motif is strictly conserved;130 however, only the Tyr residue involved in the crosslink is conserved among all the sequences.

The proposed mechanism of CDO indicates that oxygen could react with the ferrous iron center and gain two electrons from Cys93 and Tyr157 to form hydrogen peroxide. 135 This process seems like a regulation path of CDO. The intermediate of the CDO with its primary substrate cysteine has been trapped in the crystal structure by soaking the protein crystal with 5 mM cysteine containing mother liquid.114 The intermediate shows that the cysteine binds to the iron center bidentately through the sulfur and the nitrogen. The minor leaking of oxygen to the crystal results in the oxygen binding the ferrous center and form a three member ring with the sulfur from substrate and the iron center through the proximal oxygen. This phenomenon suggests that the two oxygen atoms are most likely inserted to the substrate sulfur spontaneously instead of one by one, which further implies that there is no high-valence Fe(IV) species formed as what has been trapped in other non-heme iron dependent enzyme like TauD.139

Table 6 Metal contents of copper reconstituted enzymes. Protein Cu content Fe content Cu-hCDO 0.977 0.03 Cu-hADO 0.191 0.01

3-His metal binding motif is not a strict iron motif. ADO was active with other metal.

Although the first crystal structure of CDO was solved with nickel substitution, CDO has been

reported not active with other metals. In this study, we will examine the metal selectivity and

substrate specificity of CDO. The apo hCDO and hADO were prepared as described before. The

reconstituted hCDO and hADO were then checked the metal content by ICP-OES. As shown in

table 4, the copper reconstitution of hCDO recovered 90% of the metal cofactor, whereas hADO

only adopted 10% copper. After switching the native iron with Cu(II), CDO lost the activity to-

80 ward its native substrate cysteine. The activity detection with two cysteine homologues cysteamine and homocysteine showed that CDO gained the activity toward cysteamine and homocysteine (Table 7).

Table 7 The kinetic parameters table of hCDO and hADO versus three substrates

app app app Km Km Km app app app k K k (cys) (cysteamine) (Hcy) cat m cat mM mM mM Min -1 X10 3 M-1s-1 as-isolated hCDO 0.31± 0.3 190 ± 7 10.4 3.1±0.3 102 0.55 (Ref) Cu(II)-hCDO 0.33 ± 0.1 50 ± 4 2.61 3 ± 1 40 ± 7 0.27 Cu(II)-hADO 1 ± 0.1 3 ± 1 0.05 1.2 ± 0.2 0.87 ± 0.1 0.01

This experiment revealed that CDO active site property is changed when the metal core is switch from iron to copper. The as-isolated CDO was found not active with other metals or substrate homologues. After metal switch, the enzyme lost the activity of its native substrate, instead gained new activities. Thus, in CDO, the metal plays an important role on the substrate specificity. The active site crystal structure of the CDO shows that, cysteine is bounded to the iron center through N and S the distance of which is about 2.9 Å. Two Arg residues stabilize the substrate through hydrogen bonding with the carboxyl group. Ferrous iron has six-coordination that are taken by 3-His metal binding motif, primary substrate and oxygen in CDO. Cu(II) has less coordination for the instance lost the activity of cysteine. The crystal structure of cysteamine shows a linear form of the molecule with N and S 4.0 Å apart from each other (3Q1L). Thus, the oxidation of cysteamine doesn’t need the sixth coordination of the metal for the N binding. The distance of the N and S in homocysteine is 5.2 Å (3BOF), which is even longer. So the structure of both cysteamine and homocysteine abolish the N ligation to the metal center thus enable the

81 metal with less coordination active. Homocysteine has longer side change compared to the cysteine and cysteamine thus the hydrogen network could be disturbed affecting the function of the second sphere residues around the active pocket, which results lower catalytic efficiency compared to cysteamine cysteine. Another possibility for Cu(II)-CDO activity is that the second sphere residues that are involved in the activity were altered, which results in breaking the substrate specificity restriction. To further confirm the proposals, crystallization of Cu(II)-CDO with substrate/homologue soaking is needed to be pursued.

82 5 THE STRUCTURE AND FUNCTION STUDY OF THE SPECOND SPHERE

HISTIDINE IN α-AMINO-β-CATBOXYMUCONATE-ε-SEMIALDEHYDE DECAR-

BOXYLASE

The previously reported crystal structures of α-amino-β-carboxymuconate-ε- semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His) 3(Asp)(OH 2) active

site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in

ACMSD from Pseudomonas fluorescences (Pf ACMSD). Site directed mutagenesis of His228 to

tyrosine and glycine in the present study results in complete or significant loss of activity. Metal

analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue

plays a role in metal selectivity of the protein. As-isolated H228Y displays a blue color, which is

not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the

blue color originates from Fe(III)-tyrosinate ligand-to-metal-charge-transfer (LMCT). Co(II)-

substituted H228Y ACMSD is brown in color and exhibits an EPR spectrum showing a high-

spin Co(II) center with a well-resolved 59 Co (I = 7/2) eight-line hyperfine splitting pattern. The

X-ray crystal structures of the as-isolated Fe-H228Y (2.8 Å), Co- (2.4 Å) and Zn-substituted

H228Y (2.0 Å resolution) support the spectroscopic assignment of metal ligation of the Tyr228

residue. The crystal structure of Zn-H228G (2.6 Å) was also solved. These four structures show

that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and

Zn-H228G) or disrupted (Zn-H228Y) in response to His228 mutation. Together, these results

highlight the importance of His228 for Pf ACMSD’s metal specificity as well as maintaining a

water molecule as ligand of the metal center. His228 is thus proposed to play a role in activating

the metal-bound water ligand for subsequent nucleophilic attack on the substrate.

83 5.1 Introduction

Histidine, an essential amino acid, is found at the active site of a myriad of enzymes. The

imidazole side chain of histidine can serve as a coordinating ligand in metalloproteins and in

many cases is a catalytically important component in the active sites of enzymes. The p Ka of the imidazolium ion permits significant concentrations of both acidic and basic forms near neutral pH, making it a commonly found general acid-base catalyst in enzymes. 140 The participation of

histidine in catalysis has also been illustrated in the well-studied mechanisms proposed for mem-

bers of the amidohydrolase superfamily. 141 The best-characterized members of the

amidohydrolase superfamily share a common catalytic mechanism by which a metal-bound wa-

ter is proposed to be activated by an active site base to yield a metal-hydroxo species. Subse-

quently, the hydroxide attacks the substrate bearing the amide or other functional groups at the

carbon or phosphorus center to form a tetrahedral carbon or pentacoordinate phosphorus inter-

mediate on the substrate. 141a The collapse of the substrate-based intermediate leads to the hydrolytic products.

The enzyme α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) catalyzes the decarboxylation of its substrate ACMS to form 2-aminomuconate semialdehyde product (AMS). It is the first enzyme known to perform an O 2-independent nonoxidative decarboxylation with d-block divalent metal cofactors such as zinc, cobalt, or iron. 142 ACMSD is pro-

posed to be a prototypical member of a new protein subfamily in the amidohydrolase superfami-

ly representing a novel nonhydrolytic C-C bond breaking activity. 41b, 143 Figure 39 outlines a

working mechanism of the ACMSD catalytic cycle.

ACMSD plays an important role in two distinct metabolic pathways, the mammalian

kynurenine pathway for tryptophan degradation 144 and the bacterial 2-nitrobenzoic acid pathway

for biodegradation of nitroaromatic compounds. 145 In both pathways, ACMSD controls the final

84 fate of the metabolites by competing with a slow spontaneous reaction that produces the

excitotoxin quinolinic acid (QA) and directs the metabolic flux away from QA to energy produc-

tion. 146 QA is an endogenous selective agonist of N-methyl-D-aspartate receptors. 147 It modulates

neurotransmission and mediates immune tolerance. Reduced activity of ACMSD can lead to ab-

normal QA concentration in body fluids, which has been linked to numerous diseases, including

diabetes, 146 neuropsychiatric diseases, 148 neurodegenerative disorders such as Alzheimer’s and

Huntington’s diseases, 149 stroke, and epilepsy. 147, 150 Therefore, the results of mechanistic study on ACMSD have significant medical implications.

Although ACMSD was discovered more than 55 years ago, 151 little was known about the catalytic mechanism until our recent collaborative studies of Pseudomonas fluorescences

ACMSD ( Pf ACMSD) 142 unmasked its metal cofactor and determined its three dimensional structure. 81 The as-isolated enzyme contains a zinc ion 81 and it works equally well when substituted with a cobalt(II) ion. 142-143 The decarboxylation is rapid (> 500 s -1); the product release from the active site is the rate-limiting step. 152 Recently, the crystal structure of human ACMSD was reported with a bound inhibitor 1,3-dihydroxyacetonephosphate. 153 The human ACMSD and

Pf ACMSD present a nearly identical overall structure consisting of an ( α/β)8-barrel core and a five-coordinate protein-bound zinc ion. 81, 153

The metal ion is bound to the enzyme by three histidine residues (His9, His11, and

His177), one aspartic acid residue (Asp294), and one water ligand. A nearby histidine from β- strand 6, i.e . His228 in Pf ACMSD residue numbering, is located in the secondary sphere and is part of a hydrogen-bonded system that appears to facilitate deprotonation of the water ligated to the catalytic zinc (Figure 40). A comprehensive biochemical, spectroscopic and structural study presented in this work suggests that His228 is a major determinant of metal preference in

85 Pf ACMSD and that it plays an important role in maintaining the hydrolytic water ligand necessary for the decarboxylase chemistry in this enzyme.

Figure 39 The proposed catalytic role of His228 in ACMSD reaction

5.2 Experimental procedures

Site-Directed Mutagenesis . The plasmid containing His-tagged ACMSD from Pseudo- monas fluorescens was used as a template for construction of all of the mutants. 143 The forward

primers used in the site-directed mutagenesis are 5’-

CAAGATCTGTTTCGGGggTGGTGGGGGAAGTTTCG-3’ for H228G and 5’-

CAAGATCTGTTTCGGGtATGGTGGGGGAAGTTTCG-3’ for H228Y. ACMSD mutants were

constructed by the PCR overlap extension mutagenesis technique. 154 The insertion of each con-

struct was verified by DNA sequencing to ensure that base alterations were introduced correctly

as expected and with no undesired changes. After sequencing, the positive clone was used for

overexpression of these mutants in E. coli BL21(DE3).

Bacterial Growth and Protein Preparation . The expression and purification of WT

Pf ACMSD and its mutants were the same as described previously. 143 Apo H228G was prepared

86 by EDTA treatment; Zn(II) and Co(II) containing H228G proteins were prepared by metal re-

constitution reaction as previously described. 142 Because iron cannot be removed from as-

isolated H228Y by high concentration EDTA, Zn(II)- and Co(II)-H228Y proteins were obtained

by substituting 0.1 mM CoCl 2 or ZnCl 2 to M9 medium prior to the addition of IPTG for induc- tion during cell culture. The protein was purified in the presence of 0.1 mM CoCl 2 or ZnCl 2 during affinity chromatographic step by using Co(II)- and Zn(II)-charged IMAC columns, respectively.

Metal Analysis . The metal content of the as-isolated ACMSD from LB was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) using a Spectro Gene- sis spectrometer (Spectro Analytical Instruments). Assays were performed in triplicate for the mutants the same day as the assay samples of the wild-type (WT) enzyme. Standard curves were prepared using a transition element standard mixture (CCS-6) from Inorganic Ventures. Error

(standard deviation) within three measurements of each sample was less than 1%, while error between different ACMSD preparations was within 4%. Metal content is reported per ACMSD monomer. Protein concentration was measured using Coomassie Plus protein assay reagent

(Pierce) according to the manufacturer's instruction. Molar concentration of Pf ACMSD monomer was determined by using 39 kDa as the molecular weight. 142

Circular Dichroism Spectroscopy. The CD spectra (190 - 250 nm) of ACMSD and mutant proteins were acquired on a JASCO J-810 spectropolarimeter (JASCO, Easton, MD, USA) at ambient temperature. In each measurement, a protein sample (10 M) was placed in a 1-mm path length quartz cell in 0.05 M potassium phosphate, pH 7.5. All spectra were the average of ten scans with a scan rate of 50 nm min −1 .

Preparation of ACMS and Enzyme Activity Assay. ACMS was generated from 3-hydroxyanthranilic acid using 3-hydroxyanthranilate 3,4-dioxygenase containing no free transient metal

87 ion as reported previously. 143 Specific activities of ACMSD proteins were measured in triplicate at room temperature on an Agilent 8453 diode-array spectrophotometer by monitoring the absorbance of ACMS at 360 nm as described previously. 143

Electronic Spectroscopy. UV-Vis absorption spectra were obtained in 25 mM HEPES-

NaOH buffer, pH 7.0, and 5% glycerol at room temperature on a Cary 50 spectrophotometer.

Reduced Fe(II)-H228Y for UV-Vis and resonance Raman experiments was prepared by adding

13 L 30 mM sodium dithionite or L-ascorbate (~ 2 equivalents per Fe) to 150 L 1.4 mM

Fe(III)-H228Y in an 1-mm path length cuvette.

Quinone-based NBT Staining. Purified proteins were first electrophoresized on a 12%

SDS–PAGE gel and then transferred onto a nitrocellulose membrane using the Mini Trans-Blot

Cell Assembly (Bio-Rad). The transblotting was conducted in the transfer buffer (25 mM Tris-

HCl, 192 mM glycine, and 20% methanol) at 100 V for 1 h. The proteins were temporarily visu-

alized by 0.1% ponceau S in 5% acetic acid. 155 After the temporary stains were removed by

washing with water, the protein-containing nitrocellulose membrane was immersed in a solution

of 0.24 mM NBT and 2 M potassium glycinate, pH 10, in the dark for 45 min to visualize the

quinone-containing protein band. 156 This method was designed to identify quinonoid compounds,

such as DOPA, and is a convenient assay for quinone-containing proteins.157 Purified methyla-

mine dehydrogenase (MADH) from Paracoccus denitricans (a generous gift from Dr. Victor L.

Davidson), a protein consisting of two large subunits and two small -subunits with a trypto-

phan tryptophyquinone prosthetic group in each of its small subunit, 158 was used as both of a

positive and a negative control in Ponceau S staining and NBT staining experiments.

Electronic Paramagnetic Resonance (EPR) Spectroscopy . HEPES buffer (25 mM, pH

7.0) containing 10% glycerol was used in the EPR sample preparations. X-band EPR spectra

were obtained in perpendicular mode on a Bruker (Billerica, MA) EMX spectrometer at 100-kHz

88 modulation frequency using a 4119HS high-sensitivity resonator. Low temperature was main-

tained with an ITC503S temperature controller, an ESR910 liquid helium cryostat, and a

LLT650/13 liquid helium transfer tube (Oxford Instruments, Concord, MA). Measurements were

conducted by keeping the frequency of the electromagnetic radiation constant while the magnetic

field was swept.

Raman Spectroscopy . Resonance Raman spectra were collected on an Acton AM-506

spectrophotometer (1200 groove rating) using Kaiser Optical Systems holographic supernotch

-1 filters and a Princeton Instruments liquid N 2-cooled CCD detector (LN-1100PB) with 4 cm spectral resolution. HEPES buffer (25 mM, pH 7.0) containing 5% glycerol was used in the sample preparations. Spectra were collected at 100 mW laser power using a Spectra Physics 1060-

KR-V krypton ion laser (568.2 and 647.1 nm) or a 2030-15 argon ion laser (488.0 and 514.5 nm). Raman frequencies were referenced to indene standard with an accuracy of ± 1 cm -1. Ra- man spectra were collected at 4 °C by 90° scattering from a flat bottomed NMR tube with 30 –

60 minutes to collect each window of spectra. The full spectra were collected in two overlapping windows; the florescent background was subtracted before normalizing spectra at nonresonance enhanced protein phenylalanine ring vibration at 1004 cm -1 and splicing normalized spectra to-

gether. Baseline corrections (polynomial fits) were applied using Gram/32 Spectral Notebook

(ThermoGalactic). Excitation profiles were constructed by comparing peak intensities of reso-

nance enhanced bands of normalized spectra to nonresonance enhanced vibration at 1004 cm -1.

Difference resonance Raman spectra of Fe(III)-H228Y were obtained by subtracting the spec-

trum of WT Zn-ACMSD both normalized at 1004 cm -1 vibration followed by a base line correc-

tion.

X-ray Data Collection and Crystallographic Refinement. Co(II)-H228Y, Zn(II)-H228Y

and Zn(II)-H228G ACMSD were crystallized following the condition previously established for

89 WT ACMSD by hanging drop vapor diffusion in VDX plates (Hampton Research). Single crys-

tals suitable for X-ray data collection were obtained from drops assembled with 1 L protein so-

lution layered with 1 L reservoir solution containing 0.1 M Tris-Hcl (pH 8.75), 0.2 M MgCl 2,

and 15% PEG 5000. Reservoir solution for Fe(III)-H228Y was modified and contained 0.1 M

Tris (pH 7.0), 0.2 M MgCl 2, and 17% PEG 5000. Crystals were frozen in liquid nitrogen after

being dipped into the cryoprotectant solution that contained 30% of glycerol or ethylene glycol

in the mother liquid. X-ray diffraction data were collected at SER-CAT beamline 22-ID or 22-

BM of the Advanced Photon Source (APS), Argonne National Laboratory, Argonne, IL. Data

were collected at 100 K using a beam size matching the dimensions of the largest crystal face.

The data were processed with HKL2000. 96 Structure solutions were obtained by molecular re-

placement using MolRep 97 from the CCP4i program suite 98 with the entire WT Co(II)-ACMSD structure (PDB entry 2HBX) for Co(II)/Fe(III)-H228Y ACMSD and WT Zn(II)-ACMSD structure (PDB entry 2HBV) for Zn(II)-H228Y/Zn(II)-H228G ACMSD as the search models. Re- finement was carried out using REFMAC 99 in the CCP4i program suite 98 for Fe(III)- and Co(II)-

H228Y and PHENIX software for Zn(II)-H228Y and Zn(II)-H228G, and model-building was

carried out in COOT. 100 Restrained refinement was carried out using no distance restraints between the metal center and its ligands. Residue Tyr228 and Gly228 were well-ordered and added to the model based on the 2 Fo-Fc and Fo-Fc electron density maps. Refinement was assessed as complete when the Fo-Fc electron density contained only noise.

5.3 Results

5.3.1 Biochemical properties of the His228 mutants

To probe its role in the enzyme mechanism, His228 was mutated to tyrosine, which also has a ring structure and is capable of hydrogen bonding, and glycine, which effectively deletes the side chain and thus eliminates hydrogen bonding. Both variants were expressed as soluble

90 proteins. Surprisingly, the H228Y mutant purified from cells grown in LB medium exhibits a

blue chromophore while wild-type (WT) Zn-ACMSD is colorless (Figure 41). Metal analysis of

the blue H228Y-ACMSD by ICP-OES spectroscopy showed the enzyme contained 0.51 ±

0.013% Fe, 0.08 ± 0.003 Zn and 0.001 ± 0.0004 Cu ions per polypeptide chain, whereas the

wild-type enzyme prepared under the same experimental conditions contained only zinc. 81 Like- wise, the as-isolated H228G mutant contained mainly Fe ion (0.32 ± 0.001 per polypeptide chain) and trace amount of zinc. A sharp ferric EPR signal is seen at g = 4.27 from the two as- isolated His228 mutants (not shown). These results suggest His228 is an important determinant of metal selectivity in this enzyme.

Figure 40 The active site of ACMSD. (A) as-isolated Zn-pf ACMSD (Protein Data Bank entry: 2HBV), 81 (B) Co(II)-substituted pf ACMSD (2HBX), 81 and (C) an inhibitor-bound structure of human ACMSD (2WM1). 153

The as-isolated H228G and H228Y were catalytically inactive regardless of whether the Fe ion

is in the ferrous or ferric oxidation state. Zinc- and cobalt-substituted variants were also tested.

H228Y was inactive in both Zn(II) and Co(II) forms, as was Zn(II)-H228G. These results sug-

gest that His228 is essential for the enzyme catalytic activity.

91 5.3.2 The blue color in H228Y is not due to formation of a metal-bound

dihydroxyphenylalanine

Two possibilities were considered for the origin of the blue chromophore of the as-

isolated Fe-containing H228Y. The first option is an oxygenated Tyr may be produced by post-

translational modification resulting from oxygen activation by the redox active iron. Examples

can be found for a number of non-heme iron enzymes. 157, 159 However, this option is not support-

ed by a quinone-based staining assay 157 using reversible ponceau S staining of a nitrocellulose membrane. Alternatively, it could be due to the coordination of Tyr228 to the active site metal, as tyrosinate-metal interactions have been observed in many other enzymes, such as purple acid phosphatases, 160 phospholipase D, 161 and catalases. 162

Figure 41 UV–vis spectra of Fe-H228Y. As-isolated Fe(III)-H228Y protein (dark blue), its reduced form (black), and the difference (red) showing two absorptions at 370 nm (3000 M –1 cm –1) and 595 nm (1255 M –1 cm –1). Photograph of the as-isolated Fe(III)-H228Y (right) and reduced Fe(II)-H228Y (left) are shown in the inset.

92 5.3.3 Resonance Raman characterization suggests an Fe(III)-tyrosinate chromophore in as-

isolated Fe-H228Y

The blue Fe-H228Y mutant exhibits two UV-Vis absorption bands at 370 and 595 nm,

with respective extinction coefficients of 3000 and 1300 M -1 cm -1 (Figure 42). While the 370 nm

feature is a common spectral characteristic of the Fe(III)-bound nonheme Fe proteins, the blue

chromophore at 595 nm is within the range of the Fe(III)-tyrosinate LMCT band with an absorp-

tion maximum that is typically between 410 – 600 nm (1000 – 2500 M -1cm -1 per phenolate lig-

and). 163 In contrast, an iron(III)-catecholate LMCT chromophore typically exhibits two lower

energy bands between 400 – 580 nm and 550 – 900 nm (2000 – 2500 M -1cm -1), these are not pre-

sent in Figure 42. The blue chromophore vanished upon the addition of sodium dithionite or L-

ascorbate to the protein, suggesting that reduction of the Fe(III) metal center to Fe(II) is linked

with the loss of the intense ligand-to-metal charge-transfer (LMCT) transition.

Figure 42A shows resonance Raman spectra of the as-isolated blue Fe-H228Y acquired

with 568.2 nm laser excitation using 90 ° scattering geometry compared to the spectra of reduced

Fe-H228Y, WT Zn-ACMSD and the buffer (25 mM HEPES pH 7.0 with 5% glycerol by vol-

ume) collected under the same conditions. Vibrations associated with the buffer solution and pro-

tein were subtracted by taking the difference spectra of the as-isolated blue Fe-H228Y and WT

Zn-ACMSD after normalizing both spectra by scaling to non-resonance-enhanced protein phe-

nylalanine ring vibration at 1004 cm -1 (Figure 42B). The difference spectrum shows resonance-

enhanced vibrations at 573, 792, 873, 1164, 1290, 1503, and 1604 cm -1. The excitation profile of the 1164, 1290, 1503, and 1604 cm -1 features shown in Figure 45C illustrates maximum en- hancement of these vibrations near the absorption maximum of the blue chromophore at 595 nm, while the 573, 792, and 873 cm -1 vibrations are more enhanced at 647.1 nm. The spectrum of the

93 reduced form of the colorless Fe(II)-H228Y does not exhibit any resonance-enhanced vibrations

but closely resembles the WT Zn-ACMSD (Figure 42A).

The resonance enhanced vibrations of Fe(III)-H228Y at 573, 1164, 1290, 1503, and 1604

cm -1 are collectively regarded as the Raman signature for a Fe(III)-tyrosinate chromophore (Ta-

ble 8). 163-164 These vibrations are distinct from those expected for a Fe(III)-catecholate

chromophore, which has characteristic vibrations observed at 1270, 1320, 1425, and 1475 cm -1

(Table 8). 165 By analogy to previous work on nonheme Fe proteins the 573 cm -1 can be assigned

to the Fe-(O) Tyr vibration, while the higher frequency vibrations are associated with aromatic

ring modes. The 792 and 873 cm -1 vibrations may originate from a Fermi doublet formed by the

mixing of the fundamental υ1 symmetric ring-breathing vibrational mode and first overtone υ16a

The two peaks observed for the blue Fe-H228Y have a much larger splitting of 81 cm -1.

An even larger splitting has been observed for porcine uteroferrin (803 and 873 cm -1), while the

167 υ16a fundamental was not detected. The large splitting of tyrosine Fermi doublet was rational- ized as a result of strong covalence of the Fe(III)-tyrosinate metal ligand bond based on the fact that it was resonance enhanced and that its center of gravity (~ 838 cm -1) is similar to that of L- tyrosine (~ 824 cm -1). 167a Upon 647.1 nm laser excitation of the blue Fe-H288Y, a weak peak is

-1 observed at 418 cm that may be assigned to the fundamental υ16a vibration.

Fe(III)-tyrosinate LMCT bands in the visible region typically range from 410–600 nm where the energy of the LMCT transition can be correlated with the Lewis acidity and the

Fe(III/II) redox potential of the iron center and can give additional information about the nature of the other coordinating ligands. 163 The low energy of the Fe(III)-tyrosinate LMCT band of Fe-

H228Y at 595 nm and the observation that Fe-H228Y is easily reduced by mild reducing agents like L-ascorbate are consistent with a Lewis acidic metal center with weak field ligands. 164a Post- translationally modified blue (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) has a similarly

94 low-energy Fe-tyrosinate LMCT band at 595 nm. 164a, 166 From its crystal structure, 170 unmodified

HPPD has an iron center coordinated by two histidines and one glutamate, but no tyrosinate lig- and. However it does have several phenylalanine amino acid residues in the second sphere, one of which has been proposed to be self-hydroxylated to form the tyrosinate ligand in blue-

HPPD. 165e, 170

Figure 42 Resonance Raman spectra of ACMSD. (A) Resonance Raman spectrum with 568.2 nm laser excitation of (a) as-isolated blue Fe(III)-H228Y, (b) native Zn-ACMSD, (c) Fe(II)-H228Y ACMSD, and (d) Fe(III)-H228Y ACMSD. The buffer used for these proteins was 25 mM HEPES pH 7.0 with 5% glycerol by volume. (B) Difference resonance Raman spectra of Fe(III)-H228, obtained with 488.0, 514.5, 568.2 647.1 nm laser lines, respectively, minus the spectrum of wild-type Zn(II)-ACMSD. The spectra are normalized to protein phenylalanine ring vibration at 1004 cm -1 before subtraction. (C) Excitation profiles for resonance enhanced bands compared to the extinction coefficient of Fe(III)-H228Y blue chromophore and colorless reduced Fe(II)-H228Y obtained using 488.0, 514.5, 568.2 and 647.1 nm laser lines.

96 Table 8 Resonance Raman vibrations and LMCT bands of nonheme iron protein and model complexes with Fe(III)-phenolate and Fe(III)-catecholate chromophores. Complexa λmax (nm) (ε, M- 1cm-1) Resonance Raman Vibrations (cm-1) Ref Fe(III)-phenolate complexes H228Y-Fe(III)- 370(3000), this ACMSD 595(1255) 573 792 873 1164 1290 1503 1604 work Fe(II)-HPPD + O2 595(2600) 583 751 881 1171 1290 1502 1600 164a, 166 167

Porcine uteroferrin 545(1000) 575 803 873 1173 1293 1504 1607 [Fe(salhis)2]ClO4 530(4100) 1159 1337 1476 1625 164f 1132 1310 1452 1605 Fe(III)- serotransferrin 470(2500) 759 828 1174 1288 1508 1613 164i, 164l Fe(III)-lactoferrin 465(2070) 1170 1272 1500 1604 164j , k

Fe(III)- ovotransferrin 465(2000) 759 860 1173 1264 1501 1603 164d Catechol 1,2- 450(3000– dioxygenase 4000) 757 872 1175 1289 1506 1604 164f, g Protocatechuate 435(3000), 3,4-dioxygenase 525 592 756 826 1172 1254 1506 1604 164b, 164e 854 1180 1266 [Fe(salen)(OC6H- 4-CH3)] 410 568 1168 1272 1501 1603 163, 164h Fe(III)-catecholate complexes

Fe(III)TyrH- 415(1700), dopamine 695(2000) 528 592 631 1275 1320 1425 1475 165d 420(1500), Blue PMI 680(2100) 591 631 1266 1330 1428 1482 165b Fe(III)-MndD- DOPA 675(750) 530 569 646 666 1273 1318 1423 1464 168 586 Fe(II)TauD-αKG +O2 550(700) 544 580 623 644 1261 1314 1425 1475 165c, 169

aAbbreviations used in this table: ACMSD, α-Amino-β-carboxymuconate-ε-semialdehyde decarboxylase from Pseudomonas fluorescens ; salhis, N-[2-(4-imidazolyl)ethyl]salicylaldimine; salen, N,N'-ethylenebis(salicy1idenamine) dianion; MndD, homoprotocatechuase 2,3-dioxygenase from Arthrobacter globiformis ; DOPA, dihydroxyphenylalanine; PMI, phosphomannose isomerase from Candida albicans expressed in Escherichia coli ; TauD taurine /α-ketoglutarate dioxygenase from Escherichia coli ; HPPD, (4-hydroxyphenyl)pyruvate dioxygenase from Pseu- domonas fluorescens ; α-KG, α-ketoglutarate.

Based on the similar energies of their LMCT bands, blue Fe(III)-H228Y and the self-

hydroxylated HPPD are likely to have comparable ligand environments with two or three

histidine residues and a carboxylate group from either an aspartate or glutamate. Along with the

fact that H228Y no longer picks up Zn(II), the Fe(III) is likely to be coordinated by His9, His11,

His177, Asp294 and Tyr228 residues in the ACMSD active site.

5.3.4 Spectroscopic characterization of Co(II)-substituted H228Y

Co(II)-H228Y was obtained by adding supplemental CoCl 2 to the M9 minimum growth

medium and purification buffer (see experimental procedures). ICP-OES spectroscopic analysis

confirmed that H228Y isolated with supplemented cobalt contains 0.98 ± 0.02 cobalt and 0.05 ±

0.03 iron per protein monomer. The cobalt-substituted H228Y displays a brown chromophore

that is darker than the color of the WT Co(II)-substituted ACMSD (Figure 43A), The brown

chromophore originates from forbidden metal d-d transitions,. the intensities of which can be

correlated to the metal coordination number.171 Decreasing the coordination number increases the intensity of the d-d bands due to greater p-d orbital mixing. Typically, six-coordinate high- spin Co(II) complexes have extinction coefficients of less than 50 M -1cm -1 above 500 nm, while five- and four-coordinate complexes have values between 50 – 250 M -1 cm -1 and greater than

300 M -1cm -1, respectively. 172 The visible spectrum of the brown Co(II)-substituted H228Y is similar to that of WT Co(II)-ACMSD above 500 nm (Figure 43A), consistent with a 5- coordinate Co(II) center. 173 However, the spectrum below 500 nm is distinct from that of the

wild-type protein (Figure 43A). The absorption peaks at 355 (680 ± 20 cm -1M-1) and 420 nm

(430 ± 10 cm -1M-1) in WT ACMSD are respectively red shifted to 380 (590 ± 30 cm -1M-1) and

458 (490 ± 20 cm -1M-1) in the Co(II)-substituted H228Y. These differences indicate that the

98 Co(II) center in the H228Y mutant is in a weaker ligand field environment than in WT Co(II)-

substituted ACMSD. This observation is consistent with the substitution of a water ligand by a

stronger π-donating ligand in Co(II)-substituted H228Y due to Tyr228 ligation to the metal as suggested by resonance Raman study for Fe-H228Y in as-isolated H228Y protein.

Electron paramagnetic resonance (EPR) spectroscopy was employed to characterize the electronic structure of the metal center in Co(II)-substituted H228Y. The EPR spectrum of Co-

H228Y exhibits an S = 3/2 spectrum with a well-resolved eight-line 59 Co hyperfine interaction

pattern associated with the low-field feature (Figure 43B), consistent with a high-spin Co(II) ion

bound specifically to the protein.

Figure 43 The UV-Vis and EPR spectra of Co(II)-substituted ACMSD. (A) UV–vis spectra of Co(II)-H228Y ACMSD (black) and Co(II)-substituted WT ACMSD (red). Photograph of the as-isolated Co-bound WT (left) and Co-H228Y (right) at the same protein concentrations are shown in the inset. (B) EPR spectra of Co(II)-H228Y ACMSD obtained at a 3 G modulation, 0.25 mW microwave power, and 10 K. The asterisk indicates a g = 4.27 signal from a small amount of Fe ion present in the sample due to a change in the metal preference of H228Y.

An Fe(III) EPR signal is also present at g = 4.27, showing that a minor fraction of protein

still binds iron even under the cobalt-rich conditions. The well-resolved 59 Co hyperfine structure

99 of Co-H228Y is observable even at 10 – 20 K, but only a featureless cobalt signal can be ob-

served in WT Co-ACMSD at this temperature. 142 The g value of the high-field feature of the Co-

H228Y mutant is also different from that of the WT protein, i.e ., 2.343 versus 2.613, indicating that the ligand geometry around the metal ion in H228Y is different from that of the WT enzyme. 142 The optical and EPR data suggest that the high-spin cobalt center in the brown H228Y is distinct from the cobalt center in the wild-type enzyme. The spectral features are most reminiscent of high-spin five-coordinate cobalt centers with square pyramidal geometry, such as that found in inhibitor derivatives of cobalt carbonic anhydrase. 174

5.3.5 X-ray crystal structure

While the spectroscopic data strongly suggest a tyrosinate ligation to Fe in the as-isolated

H228Y protein, it remains unclear that if Tyr228 is a metal ligand in Co(II)-substituted H228Y.

The zinc-containing mutants do not have chromophore or EPR features. In order to observe directly what changes at the enzyme active site arise from the point mutation and metal substitution, we obtained single crystals from the as-isolated Fe(III)-H228Y, Co(II)-substituted H228Y,

Zn(II)-substituted H228Y, and Zn(II)-reconstituted H228G proteins. The X-ray crystal structures of Fe(III)-H228Y (2.8 Å, PDB entry: 4ERG), Co(II)-H228Y (2.4 Å resolution, PDB entry:

4ERA), and Zn(II)-H228G (2.6 Å, PDB entry: 4EPK) were solved in the P42212 space group, while Zn(II)-H228Y (2.0 Å, PDB entry: 4ERI) was determined in the C2 space group. Data collection and refinement statistics are presented in Table 9. Figure 44 shows the superimposition of the four structures we determined against the previously reported WT Zn(II)-ACMSD and

Co(II)-substituted ACMSD data. As previously seen in the wild-type enzyme, the mutants of pf ACMSD exhibit a homodimeric quaternary structure. The substitution of His228 with either

Tyr or Gly has little impact on the folding of the overall TIM-barrel scaffold, regardless of the metal identity. The circular dichroism spectra of these variants were nearly identical to that of the

100 WT enzyme (not shown), consistent with the structural data. In Figure 44, structural variations relative to WT ACMSD are highlighted in different colors.

Table 9 X-ray crystallography data collection and refinement statistics Data collection Fe-H228Y Co-H228Y Zn-H228Y Zn-H228G MAR300 MAR300 Detector type CCD MAR225 CCD MAR300 CCD CCD APS, Sec- APS, Sector 22- APS, Sector 22- APS, Sector Source tor 22-ID BM ID 22-ID Space group P42212 P42212 C2 P42212 a = b = a = 153.84, b = 90.43, c = a = b = 90.03, c = 48.56, c = a = b = 91.50, Unit cell lengths (Å) 167.53 167.31 110.20 c=170.12 α = β = γ = α = β = 90°, γ = α = β = γ = Unit cell angles (?) 90° α = β = γ = 90° 126.89° 90° Wavelength (Å) 1 1 1 1 Temperature (K) 100 100 100 100 50 - 2.80 (2.85 - 35 - 2.40 (2.44 - 50 - 2.00 (2.03 - 50 - 2.60 Resolution (Å) a 2.80) 2.40) 2.00) (2.64 - 2.60) Completeness (%)a 99.8 (99.8) 99.2 (94.1) 92.7 (54.7) 98.4 (68.7) Rmerge (%)a, b 10.1 (69.7) 10.1 (61.0) 6.1 (22.8) 7.7 (65.6) I/?I a 60.8 (2.5) 40.5 (2.9) 51.1 (6.0) 63.1 (1.9) Multiplicity a 24.2 (9.8) 13.9 (9.6) 6.3 (3.4) 18.6 (7.2) Refinement Resolution (Å) 2.8 2.4 2 2.6 No. reflections; working/test 17041/915 26155/1382 41014/2073 22859/1173 Rwork (%)c 19.9 21.5 20.5 20.8 Rfree (%)d 27.3 29.5 25.6 28.1 Protein atoms 5194 5194 5194 5153 Ligand atoms 2 2 3 3 Solvent sites 9 88 209 32 Average B factor (Å2) Protein 75.4 57.9 45.6 65.2 Metal ion at active site (Fe, Co, or Zn) 69.5 46.2 35.3 56.7 Mg(II) ion NA NA 65.1 80.6 Solvent 72.2 50.9 41.5 50.7 Ramachandran statisticse Preferred 93.48 92.73 94.52 91.78 Allowed (%) 5.61 5.61 4.72 7.61 Root mean square deviation Bond lengths (Å) 0.011 0.011 0.008 0.009

101 Bond angles (?) 1.299 1.464 1.098 1.204 PDB access code 4ERG 4ERA 4ERI 4EPK a Values in parentheses are for the highest resolution shell. b Rmerge = Σi |Ihkl,i - ‹Ihkl ›|/ Σhkl Σi Ihkl,i , where Ihkl,i is the observed intensity and ‹ Ihkl › is the average intensity of multiple measurements. c Rwork = Σ|| Fo|-|Fc||/ Σ|Fo|, where | Fo| is the observed structure factor amplitude, and |Fc| is the calculated structure factor amplitude. d Rfree is the R factor based on 5% of the data excluded from refinement. e Based on values attained from refinement validation options in COOT.

These structural differences are mainly observed in the previously defined mobile inse r- tion domain, which is more flexible compared with other parts of the structure. 81 Limited structural differences in a few loop regions are observed due to changes of met al ligation in the metal - substituted mutants. Therefore, we conclude that no significant change in secondary structure was introduced due to the mutation and metal substitution. The organization of amino acids res i- dues surrounding the metal center is very similar in all four structures with one exception of one of the metal ligands, Asp294, which tilts towards the imidazole ring of His9 to various degrees

(Figure 45).

Figure 44 Superimposed overall structure . WT Zn(II)-ACMSD in blue color (PDB: 2HBV), WT Co(II)-ACMSD in magentas (PDB: 2HBX), Fe(III)-H228Y ACMSD in orange (PDB: 4ERG), Co(II) -H228Y in yellow (PDB:4ERA), Zn(II)-H228Y in green (PDB: 4ERI), and Zn(II) -H228G in re in red (PDB: 4EPK). The well-overlaid structural compon ents are shown in gray color. Metal ions are represented by violet spheres. The figure was produced using PyMOL (http://www.pymol.org/ ).

102 Inspection of the metal center shows that Tyr228 is a metal ligand in all three H228Y mutant proteins (Figure 45), with Tyr-to-metal bond lengths of 2.13, 2.12, and 2.41 Å in chain A, and 2.26, 2.16, 2.44 Å in chain B in the Co-, Fe-, and Zn-H228Y structures, respectively. It would appear that the interaction between the Zn ion and the Tyr228 residue weaker than corresponding interactions with Fe and Co. Nonetheless, the angles of the Tyr228 plane, phenolic oxygen and the metal, including zinc, are well within the range of typical metal-ligand angular ori- entations (110 – 140°) reported in biological systems (130.2°, 137.3°, and 128.7° in chain A, and

129.7°, 132.5°, and 122.6° in chain B in the Co-, Fe-, and Zn-H228Y structures, respectively). 175

Hence, the prediction of Tyr228 ligation by our spectroscopic work is confirmed by these as- isolated and metal-substituted H228Y structures. It should be noted that the metal center in chain

B in the mutant structures shows a noticeable degree of disorder. This was the same as previously observed in the as-isolated and cobalt-substituted wild-type ACMSD structures (2HBV &

2HBX) 18 .

103

Figure 45 The active site structure of ACMSD and H228X mutants. (A) as-isolated Fe(III)-H228Y, (B) Co(II)-substituted H228Y, (C) Zn(II)-H228Y, and (D) Zn(II)-substituted H228G. The 2Fo-Fc electron densities are taken from chain A and coun- tered at 1.5 σ. The larger spheres represent metal ions The smaller gray sphere in panel C represents a water molecule, which is weakly bound to the zinc ion (2.44 Å) as compared to the zinc- water ligand distance of 2.04 Å of wild type enzyme (2HBV). Such a water ligand is missing from the metal center in all other H228G and H228Y structures.

The most significant structural difference with respect to the WT structure is that the water ligand is missing from the metal center in Fe(III)-H228Y, Co(II)-substituted H228Y, and

Zn(II)-reconstituted H228G proteins. Modeling of the omit maps show the electron density can only be fitted by the metal ion in these structures. However, the resolution is not sufficiently high to assign the solvent content in the Fe(III)-H228Y and Zn(II)-H228G data sets, the missing water ligand cannot be reliably concluded based on these structures. In contrast, Zn(II)-H228Y and

Co(II)-substituted H228Y structures contain reasonable amount of water molecules. The water

104 ligand is missing from the cobalt structure, whereas, the water ligand is still present in the Zn(II)- substituted H228Y structure but at a much longer distance from the metal, 2.44 Å in chain A and

2.38 Å in chain B from the zinc ion as compared to 2.05 and 2.07 Å in WT Zn-ACMSD structure. Our EPR, resonance Raman and optical data suggest a low coordination number in the

Co(II)- and Fe(III)-H2228Y metal center. Taken together, it is likely that the water ligand is missing, or dissociated, from the metal center in Co(II)- and Fe(III)-H228Y mutants. Moreover, the hydrogen bonding network that potentially activates the water ligand is significantly altered.

In the WT Zn-ACMSD crystal structure the water ligand hydrogen bonds to both His228 and

Asp294, while in the H228Y mutant the distance between Asp294 and the water ligand is elon- gated to 3.60 Å in chain A and 3.67 Å in chain B, ,compared to 2.88 and 3.33 Å in the WT Zn-

ACMSD. In the H228G structure, the metal center is still five-coordinate even though the water ligand is absent. Asp294 becomes a bidentate ligand to the metal ion. From the H228G structure and the three H228Y structures, we conclude that His228 plays an important role in metal ion recruitment and maintaining the position of the water ligand.

5.4 Discussion

His228 is a major determinant of metal ion selectivity in PfACMSD —ACMSD was thought to be a cofactor-free enzyme for about 50 years until we found many divalent transition metal ions can effectively activate it. 142 In the subsequent study, we found that the as-isolated protein contains only zinc ion. 81 However, the molecular basis of metal selectivity in proteins is generally poorly understood and the preference for zinc within the active site of ACMSD has remained a mystery prior to this work. In the present study, a point mutation of a second coordination sphere His228 residue to Gly or Tyr is described to change Pf ACMSD’s metal ion selectivity from Zn to Fe ion. In general, the primary control of metal selectivity in proteins should be the metal-binding ligand set. A single site mutation on a non-metal ligand residue changes metal

105 selectivity in a protein is a surprising result. To our knowledge, the finding described in this work for metal selection role of a second coordination sphere residue is only the second example in literature. Previously, mutation of a conserved glutamate in Escherichia coli manganese superoxide dismutase has been shown to change metal preference to Fe ion. 176 In all other documented cases the residue identified for affecting metal selectivity is a metal ligand. The lesson learned from Pf ACMSD His228 is anticipated to improve our understanding of metal ion selectivity in proteins and expand the scope of roles histidine plays in the enzyme active site.

5.4.1 The origin of blue color in H228Y

To understand the underlying reason for the loss of enzyme activity due to mutation of

His228, a spectroscopic study was performed to interrogate the metal center’s chemical and electronic structure in H228Y. The optical and NBT-staining data suggest the ligation of Tyr228 to the Fe ion in the as-isolated Fe(III)-H228Y, and this notion is further supported by our resonance

Raman results. These data also eliminate the possibility of post-translational modification of

Tyr228 to a DOPA.

5.4.2 The role of His228 in maintaining the hydrolytic water ligand for catalysis

His228 attracted our attention because it is a strictly conserved active site residue. It lies on the opposite side of the presumed substrate binding pocket. 81, 153 A previous structural study has revealed the conformational diversity of this active site residue. 81 In the present work,

His228 was mutated to tyrosine and glycine. In line with our expectations, neither of the protein variants had a measureable catalytic activity; regardless of which metal was incorporated into the active site. The biochemical and spectroscopic data suggest that the loss of catalytic activity is, in large part, due to the missing histidine that acts as an acid/base catalyst and, the loss of the water ligand. Thus, a dual role of His228 is revealed, i.e ., it stabilizes the water ligand while plays an important role in metal selectivity of the enzyme.

106 Tyrosinate ligation to the metal center was identified by resonance Raman spectroscopy and further supported by the EPR data. Fe(III)-tyrosinate LMCT bands range from 410–600 nm were the energy of the LMCT can be correlated to the Lewis acidity and the redox potential of the Fe(III) center and give additional information about the nature of the other coordinating ligands. The low energy LMCT band of H228Y-Fe(III)-ACMSD (595 nm) is suggestive of a very

Lewis acidic metal center with very weak field ligands. A low coordination number (< 6) may lead to a Lewis acidic Fe(III) and a low energy LMCT. The UV-Vis absorption spectra of both

WT and Co(II)-substituted H228Y suggest five-coordinate metal centers. Therefore, the water ligand in the wild-type enzyme is likely replaced by the Tyr228 residue in the mutant. In the crystal structures of H228Y proteins loaded with three different metal ions, Tyr228 is a metal ligand. The spectroscopic data and the crystallographic results are consistent to each other.

A water ligand is observed in all published structures of ACMSD, including Co(II)- and

Zn(II)-Pf ACMSD, as well as Zn(II)-hACMSD proteins. Such a water ligand is also present in other members of the ACMSD subfamily, including 4-oxalomesaconate hydratase, and γ- resorcylate decarboxylase. 81, 177 The structures of Fe(III)-H228Y, Co(II)-H228Y and H228G are the first structures determined that do not show a water ligand, while Zn(II)-H228Y shows a weakly bound water ligand with a longer distance to the metal center compared to the WT protein. Because of its weak binding to the Zn center, the p Ka of the water ligand in Zn(II)-H228Y may not be decreased as effectively as in the WT proteins by coordination to the zinc ion. Cobalt and zinc ions are particularly strong Lewis acids and can dramatically reduce the p Ka of bound water ligands. In a well-established example of carbonic anhydrase, the bound Zn(II) ion de-

178 creases the p Ka of its water ligand from 15.7 to 7. Catalysis ensues from this zinc activation of its bound water. His228 is strictly conserved within all known ACMSD amino acid sequences, and in all available structures of the enzyme this residue is in hydrogen bonding distance to the

107 water ligand. The primary role of this His228 is proposed as deprotonation of the metal-bound water so that a hydroxide ion can be generated as an active site nucleophile at ca. pH 6 – 7, the optimum pH range for ACMSD. Although the hydroxide attack mechanism model shown in

Scheme 1 for ACMSD is distinct from other metal-dependent decarboxylases, it shares common features with the established mechanisms in the amidohydrolase superfamily. 141a

The results described in this work are consistent with our working model of the ACMSD reaction (Fig. 39). In the working mechanistic model shown in Fig. 39, the metal-bound water molecule is deprotonated to hydroxide anion (OH -) with the assistance of His228, which is acting

as a general acid/base catalyst to deprotonate the zinc-bound water molecule for attack on the

substrate. The hydroxide ion performs a nucleophilic attack on the C2=C3 double bond of

ACMS with concomitant protonation at C3. Essentially, a water molecule is added across the

double bond with the hydroxyl group at C2 and the new proton at C3, generating a substrate-

based tetrahedral intermediate. Collapse of the tetrahedral intermediate initiates the decarboxyla-

tion and produces α-aminomuconate-ε-semialdehyde and regenerate the metal center. This decarboxylation model follows a hydrolytic mechanism consistent with the mechanistic paradigm of the amidohydrolase superfamily.

ACKNOWLEDGMENTS: We thank Dr. Victor L. Davidson for the generous gift of a control sample used in the quinone-based NBT staining, and Drs. Xiaodong Cheng and John Horton for allowing us use of the X-ray crystal screening facility at Emory University on a fee-based contract. X-ray data were collected at the Southeast Regional Collaborative Access Team (SER-

CAT) 22-ID and 22-BM beamlines at the Advanced Photon Source, Argonne National Laborato- ry. We thank Dr. Bi-Cheng Wang and the SER-CAT staff at section 22 for assistance with re- mote data collections. Use of the Advanced Photon Source was supported by the U. S. Depart-

108 ment of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-

109-Eng-38.

Supporting Information Available

The Supporting Information includes two figures. Figure S1 shows the NBT staining test and

Figure S2 shows the omit maps of the meter center in the X-ray crystal structure of His228 mutants. This material is available free of charge via the Internet at http://pubs.acs.org.

109

6 SUMMARY

ADO and CDO are the only known thiol dioxygenases in mammals, which play the central role in the sulfur metabolism pathway.179 Cysteine, cysteamine and homocysteine are three major intermediates in the sulfur metabolism. Cysteine is a precursor to glutathione and iron- sulfur clusters which both function as the reducing source in physiological conditions. 180

Cysteamine is the precursor for biosynthesis of coenzyme A. Cysteamine is used in pharmaceuti-

cal clinical trials for metabolic defect in cystinosis, radiation sickness and Huntington’s

disease. 181 The mechanistic understanding of CDO and ADO at the molecular level will contribute to the understanding of the sulfur metabolism regulation and provide the knowledge for drug design for disease treatments.

Two mechanisms have been proposed for CDO. The argument of the two is whether or not the iron center in the enzyme needs to go through high valent Fe(IV) state to insert oxygen to the substrate. To differentiate the two mechanism, we applied metal substitution in ADO to ex- change the native iron to copper and nickel which are lack the equal high valent state as Fe(IV).

The ICP-OES and the active assay results showed that copper and nickel reconstituted ADO are both active and copper ADO has even better catalytic efficient than native iron ADO. Thus we think in ADO and CDO, the metal center unlikely go through the high valent state for the catalysis. Since the structure is not available, the structural reason for the metal selectivity in ADO re- minds unrevealed.

The spectroscopic and crystallographic study on ACMSD revealed the catalytic and structural function of a second sphere residue His228. When the active site residue His228 was mutated to tyrosine, the tyrosine side chain was found to bind with the metal center in crystals.

The H228Y mutation lost the decarboxylation activity because the extra tyrosine residue re-

110 placed the metal bounded water, as the consequence no hydroxide formation could be proceeded to initiate the reaction. In most of the proposed enzymatic catalysis mechanisms, the function of the second sphere residues is very usually included. Some second sphere residues are directly involved in the catalytic mechanism such as initiating the reaction by interacting with the substrate; acting as general base/acid or indirectly contribute to the function of enzyme by stabilizing the substrate/intermediate/product through hydrogen bounding. In ACMSD, the active site histidine 228 plays two roles, which are metal selectivity and metal ligated water deprotonation.

The H228 mutation to tyrosine results in dead enzyme with ferric iron instead of the native metal zinc.

In summary, the study of the 3-His non-heme thiol dioxygenase and the unique decarboxylase not only help to solve specific mechanistic understanding problems but also help to understand the structure and function relationship of the metalloenzymes from the following three aspects: the metal core, the metal binding motif and the second sphere residues.

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