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Subject Chemistry

Paper No and Title 15: Bioinorganic Chemistry

Module No and 26: Metalloproteins Title Module Tag CHE_P15_M26

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 26: Metalloproteins

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TABLE OF CONTENTS

1. Learning Outcomes 2. Introduction 3. Function of Metalloproteins 4. Co-ordination chemistry behind metalloproteins 5. Four classes of electron transferases 5.1 Flavodoxins 5.2 Cytochromes 5.3 Iron-sulfur proteins 5.4 Blue-Copper proteins 6. Summary

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1. Learning Outcomes

After studying this module, you shall be able to

 Know the difference between simple and conjugated proteins.  Categorize conjugated proteins on the basis of prosthetic group.  Learn the role of metalloproteins.  Know the coordination chemistry behind metalloproteins.  Identify the four classes of the electron transferases: Flavadoxin, cytochromes, iron-sulfur proteins and blue copper proteins.

2. Introduction

Several types of proteins are available in nature, each of which is associated with different components and hence belong to various categories. Proteins containing only amino acid residues and no other chemical constituents are termed as simple proteins, examples include enzymes ribonuclease A and chymotrypsinogen. Whereas, proteins that contains both chemical components and amino acids are called as conjugated proteins. The non–amino acid part of a conjugated protein is commonly called its prosthetic group. Further classification of conjugated proteins is on the basis of the chemical nature of their prosthetic groups. Therefore, proteins containing lipids are termed as lipoproteins, those containing sugar groups are termed as glycoproteins, and the proteins containing metal ions are called as metalloproteins (Figure 1). A number of proteins have more than one prosthetic group. Usually the prosthetic group plays a substantial part in the protein’s biological function.

v Figure. 1 Metalloproteins

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Eversince the first X-ray crystal structure of a protein, sperm whale myoglobin, indicated the presence of an iron atom, metalloproteins drew immense attention of biochemists and chemists. Metalloproteins are responsible for catalysing important biological processes, like as photosynthesis, respiration, water oxidation, molecular oxygen reduction and nitrogen fixation and are account approximately half of all proteinsin nature. This is validated by the active-site structures of the enzymes involved, which are often reminiscent of minerals. Although metal centres form the basis of reactions, the protein matrix regulates reactivity.

3. Function of Metalloproteins

Metalloproteins are a class of biologically important macromolecules and account for nearly half of all proteins in biology. They are responsible for performing some of the most difficult yet important functions, including photosynthesis, respiration, water oxidation, oxygen transport, electron transfer, oxygenation and nitrogen fixation. These diverse functions of metalloproteins have been supposed to depend on the ligands from the amino acid, coordination structures, and protein structures in the immediate vicinity of metal ions. Huge amount of efforts have been devoted toward understanding the structure and function of metalloproteins.

It is estimated that approximately half of all proteins contain a metal and about one quarter to one third of all proteins are supposed to have metals in order to carry out their functions. It is due to the presence of the metal ions that allows metalloenzymes to perform functions which cannot be performed by the functional groups found in amino acids. Metalloproteins deal with almost all aspects related to the intracellular and extracellular metal-binding proteins, including their structures, properties and functions. The biological roles of metal cations and metal-binding proteins are limitless. Hence, metalloproteins play many different roles in cells, such as storage and transport of proteins, enzymes and signal transduction proteins.

4. Coordination chemistry behind metalloproteins

The metal ions in metalloproteins are typically coordinated to nitrogen, oxygen or sulfur centres that belong to amino acid residues of the polypeptide chain incorporated into the protein. Amongst all, some important substituents are the imidazole, thiolate and carboxylate groups in histidine, cysteinyl, and aspartate residues, respectively. Bestides this, deprotonated amides and amide carbonyl oxygen centers are the donor groups which is provided by peptide backbone. Moreover, a large number of organic cofactors also act as donors to the metal ions, such as the tetradentate N4 macrocyclic ligands incorporated inside the heme protein and inorganic ligands such as sulfide and oxide.

5. Four classes of electron transferases

In most of the biological processes, such as photosynthesis, respiration, collagen synthesis, steroid metabolism, the immune response, drug activation, neurotransmitter metabolism, and nitrogen fixation, electron-transfer reactions metalloproteins play major roles. Out of which, both

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respiration and photosynthesis are extremely significant processes as they provide most of the energy that is required to sustain life. Metalloprotein which has a single type of redox cofactor can be classified into two general groups: electron carriers and proteins involved in the transport or activation of small molecules. It is assumed that proteins that function as electron transferases typically place their prosthetic groups in a hydrophobic environment where in addition to ligand, hydrogen bonds may be provided which help to assist in stabilization of both the oxidized and the reduced forms of the cofactor. In order to minimize inner-sphere reorganization, metal-ligand bonds remain intact upon electron transfer. Many of the complex metalloenzymes containing multisite such as: cytochrome c oxidase, xanthine oxidase, and the nitrogenase FeMo protein have redox centers that function as intramolecular electron transferases.

There are four classes of electron transferases: blue copper proteins, cytochromes, flavodoxins, and iron-sulfur proteins, each of which contains many members that exhibit important structural differences.

5.1 Flavadoxins

The flavodoxins are proteins with molecular weights in the range of 8-13 kDa and are found in many species of bacteria and algae. These proteins contain an organic redox cofactor, flavin mononucleotide (FMN cofactor) that is found at one end of the protein, near the molecular surface, but only the dimethylbenzene portion of FMN is significantly exposed to the solvent. FMN can act as either a 1- or a 2-electron redox center. In solution, the semiquinone form of free FMN is unstable and disproportionate to the oxidized form called quinine and reduced form called hydroquinone (Figure 2). Hence, free FMN works as a two-electron reagent. On the other hand, FMN in flavodoxins, can function as a single-electron carrier. This is easily differentiated on the comparison of reduction potentials for free and protein-bound FMN.

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Figure 2. Flavodoxin in three oxidation states.

Evidently, the protein medium is considered to be responsible for this drastic alteration in oxidation- state stability. It was also concluded from the NMR study of the M. elsdenii flavodoxin quinone/ semiquinone and semiquinone/hydroquinone electron self-exchange rates, that the latter is approximately 300 times faster than the former, in keeping with the view that the physiologically relevant redox couple is semiquinone/ hydroquinone.

5.2 Cytochromes

Out of the four classes of the electron transferases, the cytochromes are the most thoroughly characterized class and were among the first to be identified in cellular extracts due to their unique optical properties and prominent intense absorption in the region of 410-430 nm known as the Soret band.

By definition, cytochromes are proteins containing one or more heme cofactors (Figure 3). These proteins are generally classified on the basis of heme type. There are three most commonly encountered types of heme: heme a possesses a long phytyl "tail" and is found in cytochrome c CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 26: Metalloproteins

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oxidase; heme b is found in b-type cytochromes and globins; heme c is covalently bound to c-type cytochromes via two thioether linkages.

Figure 3. Cyctochrome c

The real challenge lies in the nomenclature of these cytochromes. Some of them are designated according to the historical order of their discovery, e.g., cytochrome C2 in bacterial photosynthesis. Others are designated according to the Amax of the band in the absorption spectrum of the reduced protein (e.g., cytochrome C55]).

Of all, cytochrome c is most dominant in nature. Ambler divided these electron carriers into three classes on structural grounds. Class I cytochromes c contain axial His and Met ligands, with the heme located near the N- terminus of the protein. These proteins are globular and X-ray studies of Class I cytochromes c from a variety of eukaryotes and prokaryotes clearly demonstrated an evolutionarily preserved "cytochrome fold," with the edge of the heme solvent-exposed. Cytochromes possess positive reduction potentials (200 to 320 mV).

Class II cytochromes c (EO I ~ - 100 mV) are found in photosynthetic bacteria, where they serve an unknown function. Unlike Class I, these c-type cytochromes are high-spin where the iron is five- coordinate with an axial His ligand. The vacant axial coordination site is buried in the interior of protein. These proteins, generally referred to as cytochromes c, are four-ex-helix bundles.

Class III cytochromes c, also called cytochromes C3, contain four hemes, each ligated by two axial histidines. These proteins are found in a restricted class of sulfate-reducing bacteria and may be associated with the cytoplasmic membrane. The low molecular weights of cytochromes C3 (~14.7 kDa) require that the four hemes be much more exposed to the solvent than the hemes of other cytochromes, which may be in part responsible for their unusually negative (- 200 to - 350

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mV) reduction potentials. These proteins possess many aromatic residues and short heme-heme distances, two properties that could be responsible for their anomalously large solid-state electrical conductivity.

5.3 Iron-sulfur proteins

The iron-sulfur proteins play essential role as electron carriers in nearly all living organisms, and take part in plant photosynthesis, nitrogen fixation, steroid metabolism, and oxidative phosphorylation, as well as many other processes (Figure 4).

The simplest of all iron-sulfur proteins are the rubredoxins, which are primarily found in anaerobic bacteria. Rubredoxins are small proteins (6 kDa) and contain iron ligated to four Cys sulfurs in a distorted tetrahedral arrangement. The reduction potentials of iron-sulfur proteins are typically quite negative, indicating a stabilization of the oxidized form of the redox couple as a result of negatively charged sulfur ligands. Ferredoxins [2Fe-2S] are the other class of iron-sulfur proteins (10-20 kDa) which are found in plant chloroplasts and mammalian tissue. The structure of Spirulina platensis ferredoxin confirmed earlier suggestions, based on EPR and Mossbauer studies, that the iron atoms are present in a spin-coupled [2Fe-2S] cluster structure.

The optical spectra of all iron-sulfur proteins are very broad due to numerous overlapping charge- transfer transitions that impart red-brown-black colors to these proteins. In contrast, the EPR spectra of iron-sulfur clusters are quite distinctive, and have significant value in the redox chemistry studies of these proteins.

Four-iron clusters [4Fe-4S] are also termed as ferredoxins, found in many strains of bacteria and in most of these bacterial iron-sulfur proteins, two such clusters are present in the protein. These proteins are small with molecular weight of 6-10 kDa and have reduction potentials in the - 400 mV range. Each of these clusters contains four iron centers and four sulfides at alternate corners of a distorted cube where each iron is coordinated to three sulfides and one cysteine thiolate. The irons are strongly exchange-coupled, and the [4Fe-4S] cluster in bacterial ferredoxins is paramagnetic when reduced by one electron. The so-called "high-potential iron sulfur proteins" (HiPIPs) are found in photosynthetic bacteria, and exhibit anomalously high (~350 mV) reduction potentials. The C. vinosum HiPIP (10 kDa) structure demonstrates that HiPIPs are distinct from the [4Fe-4S] ferredoxins, and that the reduced HiPIP cluster structure is significantly distorted, as is also observed for the structure of the oxidized P. aerogenes ferredoxin. In addition, oxidized HiPIP is paramagnetic, whereas the reduced protein is EPR silent.

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Figure 4. Four types of iron-sulfur clusters with one, two, three and four iron atoms respectively.

5.4 Blue-Copper Proteins The blue copper proteins are characterized by intense S(Cys) Cu charge transfer absorption near 600 nm, an axial EPR spectrum displaying an unusually small hyperfine coupling constant, and a relatively high reduction potentia1. With few exceptions (e.g., photosynthetic organisms), their precise roles in bacterial and plant physiology remain obscure. X-ray structures of several blue copper proteins indicate that the geometry of the copper site is approximately trigonal planaras illustrated by the Alcaligenes denitrijicans azurin structure(Figure 5). In all these proteins, three ligands (one Cys, two His) bind tightly to the copper in a trigonal arrangement. Suppose, EO = 276 mV for A. denitrijicans azurin, whereas that of P. vulgaris plastocyanin is 360 mV. These differences in bond lengths are expected to stabilize copper in azurin to a greater extent than in plastocyanin, and result in a lower EO value for azurin.

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Figure 5. Azurin

6. Summary

Simple proteins contain only amino acid residues and no other chemical constituents. Conjugated proteins contain both chemical components and amino acids residues. The non–amino acid component of a conjugated protein is commonly called its prosthetic group and further classification of conjugated proteins is on the basis of the chemical nature of their prosthetic groups: proteins containing lipids are termed as lipoproteins, those containing sugar groups are termed as glycoproteins, and the proteins containing metal ions are called as metalloproteins. Metalloproteins account for nearly half of all proteins in nature and the protein metal- binding sites are responsible for catalyzing important biological processes, such as photosynthesis, respiration, water oxidation, molecular oxygen reduction and nitrogen fixation. Metalloproteins deal with almost all aspects related to the intracellular and extracellular metal-binding proteins, including their structures, properties and functions. Metalloproteins play many different roles in cells, such as storage and transport of proteins, enzymes and signal transduction proteins. The metal ions in metalloproteins are typically coordinated to nitrogen, oxygen or sulfur centres that belong to amino acid residues of the polypeptide chain incorporated into the protein. There are four classes of electron transferases: blue copper proteins, cytochromes, flavodoxins, and iron-sulfur proteins, each of which contains many members that exhibit important structural differences.

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 26: Metalloproteins