A Functional Nitric Oxide Reductase Model

A functional nitric oxide reductase model

James P. Collman*, Ying Yang, Abhishek Dey, Richard A. Decre´ au, Somdatta Ghosh, Takehiro Ohta, and Edward I. Solomon

Department of Chemistry, Stanford University, Stanford, CA 94305

Contributed by James P. Collman, September 2, 2008 (sent for review August 7, 2008)

A functional heme/nonheme nitric oxide reductase (NOR) model is presented. The fully reduced diiron compound reacts with two equivalents of NO leading to the formation of one equivalent of N2O and the bis-ferric product. NO binds to both heme Fe and nonheme Fe complexes forming individual ferrous nitrosyl species. The mixed-valence species with an oxidized heme and a reduced nonheme FeB does not show NO reduction activity. These results are consistent with a so-called ‘‘trans’’ mechanism for the reduction of NO by bacterial NOR.

functional model ͉ NO reduction ͉ N2O ͉ “trans” mechanism

Fig. 1. Schematic representation of the bimetallic active sites of NOR and itric oxide reductase (NOR) is a membrane-bound enzyme CcO. Nthat catalyzes the 2eϪ reduction of nitric oxide (NO) to nitrous oxide (N2O), an obligatory step involved in the sequen- Ϫ tial reduction of nitrate to dinitrogen known as bacterial deni- FTIR reveals a heme ferric nitrosyl band at 1,924 cm 1 that shifts trification. The active site of NOR consists of a monohistidine to 1,887 cmϪ1 when 15NO is used, whereas the EPR spectrum ligated five-coordinate heme and a trisimidazole ligated non- manifests a low-spin ferric signal that is assigned to the nonheme heme FeB. This structure strongly resembles the active site of FeB because a ferric heme nitrosyl would be EPR silent. The NO oxygen reduction enzyme-cytochrome c oxidase (CcO), which adducts of both heme Fe and FeB were obtained separately from II II II possesses a heme-a3/CuB center (Fig. 1) (1, 2). Essentially, the the reaction of NO with LFe and LZn /Fe . These NO adducts distal metal CuB in CcO is replaced by a nonheme Fe metal in were characterized with a series of spectroscopic methods in- NOR; NOR and CcO are thought to be distant relatives. cluding UV-vis, EPR, resonance Raman, FTIR, and mass spec- The dinuclear iron active site in NOR was confirmed a decade troscopy. The reaction of NO with the mixed-valence compound ago by spectroscopic studies (3). Presumably, two NO molecules LFeIII/FeII was also investigated. Our data show the formation of II III III II are turned over to give one molecule of N2O and one molecule a mixture of LFe -NO/Fe and LFe -NO/Fe -NO species, but of H2O at the diiron center with the consumption of two no N2O was detected. These results are consistent with a electrons and two protons. Although many enzyme studies of so-called ‘‘trans’’ mechanism for the reduction of NO to N2Oby NOR have been focused on the intermediate trapping and NOR. elucidation of the reaction mechanism (4–15), the details of the catalytic cycle are still unresolved because of the lack of Results structural information and uncertainty regarding short-lived Syntheses and Characterization of Dinuclear Complexes. The re- intermediates. duced diiron complex LFeII/FeII is readily synthesized by reaction In contrast to enzyme studies, synthetic biomimetic model II of LFe complex (25) with 1 equivalent of Fe(OTf)2(MeCN)2 in complexes provide a straightforward and controlled method to THF at room temperature under a N2 atmosphere. The UV-vis understand how this chemical transformation proceeds at the spectrum shows a fast and clean formation of a dinuclear product enzyme active site. However, only a few synthetic models have with shifts for both the Soret (426–424 nm) and Q bands been developed that mimic the active site of NOR; moreover, (535–530 nm) and a slightly diminished intensity in both bands these compounds either lack a proximal imidazole ligand (16, 17) (Fig. 3, solid to dotted line). The dinuclear compound LZnII/FeII or use pyridine as a replacement for the histidine ligands was synthesized by using a similar method from reaction of LZnII (18–20). No functional NOR models have been reported to date. with Fe(OTf)2(MeCN)2 [UV-vis spectra are in supporting in- Our CcO model complexes have proved to be functionally active formation (SI) Fig. S1]. The mixed-valence compound LFeIII/ for oxygen reduction reaction with minimal reactive oxygen FeII was obtained by oxidation of LFeII/FeII with one equivalent species (ROS) formation (21–24). These appear to be promising of ferrocenium tetrafluoroborate. The bis-ferric LFeIII/FeIII was NOR model candidates if the distal Cu metal is replaced by an generated by oxidation of LFeII/FeII with either two equivalents iron because the resulting diiron compound has almost all of the of ferrocenium tetrafluoroborate or with dioxygen. The struc- key components in NOR: a heme Fe with a proximal imidazole tures of these dinuclear compounds were confirmed by electro- ligand and a trisimidazole ligated nonheme Fe center. spray mass spectroscopy. In this report, we disclose the first synthetic functional NOR model LFeII/FeII (Fig. 2), which reacts with two equivalents of

NO to give one equivalent of N2O and the bis-ferric product. We Author contributions: J.P.C. and Y.Y. designed research; Y.Y., A.D., R.A.D., S.G., and T.O. have shown that NO binds to both heme Fe and FeB to form a performed research; Y.Y. contributed new reagents/analytic tools; Y.Y., A.D., R.A.D., S.G., possible bis-nitrosyl intermediate; subsequently, the two bound T.O., and E.I.S. analyzed data; and Y.Y. and A.D. wrote the paper. NO molecules are reduced to N2O by electrons from both Fe The authors declare no conﬂict of interest. centers, leaving both heme Fe and FeB in an uncoupled ferric *To whom correspondence should be addressed. E-mail: [email protected]. state. N2O has been quantitatively identified by using an enzyme, This article contains supporting information online at www.pnas.org/cgi/content/full/ nitrous oxide reductase (N2OR) that reduces N2OtoN2. The 0808606105/DCSupplemental. bis-ferric product was characterized by both FTIR and EPR: © 2008 by The National Academy of Sciences of the USA

15660–15665 ͉ PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808606105 Downloaded by guest on September 29, 2021 .(Fig. 4. UV-vis spectra of LFeII (solid line) and LFeII؉NO (dotted line II Fig. 2. Representation of the model complexes of ligand L: LFe (no MB); II II 2ϩ II II 2ϩ 2ϩ III II 3ϩ LFe /Fe (M ϭ MB ϭ Fe ); LZn /Fe (M ϭ Zn ,MB ϭ Fe ); LFe /Fe (M ϭ Fe , ϭ 2ϩ III III ϭ 3ϩ ϭ 3ϩ MB Fe ); LFe /Fe (M Fe ,MB Fe ) (metals have triﬂate counterions). reveals a well characterized S ϭ 3/2 nonheme Fe-NO signal at g ϭ 4.0 (Fig. 8, dashed line). Note that the small perturbation of the Zn-porphyrin Soret implies that the NO probably binds in the Reaction of NO with LFeII, LZnII/FeII and LFeII/FeII. LFeII ؉ NO. Addition II pocket but does not bridge the two metals. Binding of NO to FeB of purified NO to a THF solution of LFe leads to formation of leads to LZnII/FeII-NO species. Both the LFeII-NO and LZnII/ its mono-nitrosyl derivative LFe-NO. This reaction is associated FeII-NO species are stable in the solid state for a prolonged with a decreased intensity of the Soret band at 427 nm and a Q period when exposed in air. These mononitrosyl complexes were band shift from 535 to 550 nm (Fig. 4). The EPR spectrum shows also characterized by high-resolution electrospray mass spec- ϭ aS 1/2 signal (Fig. 8, solid line) typical of a six-coordinate troscopy (Figs. S2–S5). ferrous NO complex akin to those reported for the active site in LFeII/FeII؉NO. Addition of NO to the THF solution of LFeII/FeII CcO (26–30). The FTIR spectrum shows a new band at 1,630 at room temperature leads to rapid changes in the UV-vis Ϫ1 Ϫ1 cm for the LFe-NO complex that shifts to 1,600 cm with spectrum. The Soret band shifts from 424 to 423 nm, and the Q 15NO (Fig. 5, dotted and dashed lines). The resonance Raman band shifts from 530 to 550 nm (Fig. 3, dotted to dashed line). spectrum obtained by excitation with a 425-nm laser shows an FTIR spectra obtained from solid samples of the end product Ϫ Ϫ Fe-N stretch at 581 cm 1 that shifts to 545 cm 1 with 15NO exhibit a new band at 1,924 cmϪ1 that is absent in LFeII/FeII (Fig. Ϫ CHEMISTRY substitution. An Fe-NImidazole stretch is also observed at 238 9, dotted and solid lines). This band is shifted to 1,887 cm 1 when Ϫ cm 1, providing further evidence for the presence of a six- 15NO is used for the sample preparation (Fig. 9, dashed line). coordinate iron nitrosyl (Fig. 6). The 37-cmϪ115N-isotope shift is consistent with literature data LZnII/FeII؉NO. Addition of NO to LZnII/FeII in THF results in for heme ferric nitrosyls (18, 31, 32). The resonance Raman data slight blue shifts (0.5 nm for the Soret band and 1 nm for the Q on this complex show a Fe-NO stretch at 610 cmϪ1 (Fig. 10, solid band) in the UV-vis spectra (Fig. S1). The FTIR of the 14NO line) that shifts to 598 cmϪ1 upon 15NO substitution (Fig. 10, adduct of LZnII/FeII exhibits a new vibration frequency at 1,810 dotted line). The Fe-NO bending vibration of this species is cmϪ1 (Fig. 7, dotted line) that shifts to 1,774 cmϪ1 with 15NO observed at 589 cmϪ1 that shifts to 580 cmϪ1 upon 15NO substitution (Fig. 7, dashed line). These features are not ob- substitution. These values are characteristic of a ferric heme NO served in the LZnII/FeII (Fig. 7, solid line). The Fe-NO vibrations species. The EPR data of the end product show an S ϭ 1/2 signal could not be identified with resonance Raman because this (g ϭ 2.07, 2.02, 1.96) that is not perturbed by 15NO substitution region was obscured by porphyrin bands. The EPR spectrum (Fig. 8, dotted line). These data and spin integration indicate that a single low-spin FeIII is present in the product. The above spectroscopic data are consistent with the formation of LFeIII- NO/FeIII-OH (Scheme 1); the vibrational features i.e., Fe-N ϭ 610 cmϪ1 in the Raman and N-O ϭ 1,924 cmϪ1 in the FTIR are derived from a ferric heme nitrosyl that is diamagnetic (i.e., EPR

Fig. 3. UV-vis spectra of LFeII (solid line), LFeII/FeII (dotted line), and LFeII/ Fig. 5. IR spectra of LFeII (solid line) and its 14NO (dotted line) and 15NO .FeII؉NO (dashed line). (dashed line) derivatives

Collman et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 15661 Downloaded by guest on September 29, 2021 Fig. 7. IR spectra of LZnII/FeII (solid line) and its 14NO (dotted line) and 15NO Fig. 6. Resonance Raman data on LFe-14NO (solid line) and LFe-15NO (dotted (dashed line) derivatives. line). reductase activity i.e., two molecules of NO are reduced to one silent), and the S ϭ 1/2 EPR signal is derived from the nonheme molecule of N2O at the fully reduced diiron center, leaving a ferric center in the distal pocket. The assignment of this end di-ferric compound. The yield of N2O is nearly quantitative product is further buttressed by comparable FTIR, resonance within the error of the enzyme assay. A putative diferric complex Raman, and EPR (g ϭ 2.07, 2.00, 1.95) obtained by addition of formed initially after reduction of NO to N2O reacts further with NO to LFeIII/FeIII, followed by addition of an equivalent of NO, forming a Fe3ϩ-NO/Fe3ϩ-OH species as indicated by EPR, sodium methoxide (comparable to OH) (Fig. S6).† FTIR, and resonance Raman spectroscopy methods. The Ϫ1 We have used a nitrous oxide reductase enzyme (N2OR) that ␯(N-O) of our ferric heme nitrosyl is Ϸ20 cm higher than the Ϫ1 reduces N2OtoN2 to identify the formation of N2O in this ␯(N-O) of the ferric heme nitrosyl of NOR (1,904 cm ) (33) and II II Ϫ1 reaction. Samples with 1 mM LFe /Fe on addition of 3 mM NO cytochrome cbb3 oxidase (1,903 cm ) (34) but similar to the show specific activities of 43 Ϯ 5, whereas the background neutral Met Mb-NO (1,921 cmϪ1) (31, 35). The EPR spectrum activity with the same amount of NO gas is Ϸ10 Ϯ 2. This activity of the reaction product reveals a low-spin ferric signal, which is reflects an N2O concentration of 1 mM in solution, which, in this assigned to a ferric nonheme because a ferric heme nitrosyl case, implies a quantitative yield. This, in addition to the would be EPR silent (S ϭ 0). A bis-ferric compound LFeIII-NO/ vibrational and EPR data presented above, indicates that the FeIII-OMe prepared by the reaction of LFeIII/FeIII with NO and II II synthetic model complex LFe /Fe reduces two molecules of NO one equivalent of NaOMe exhibits a very similar low-spin ferric to N2O (Scheme 1). signal in the EPR. This strongly suggests that an OH ligand is bound to the ferric nonheme center (Table 1). Reaction of NO with the Mixed-Valence Compound LFeIII/FeII. Addi- III II tion of NO to the mixed-valence compound LFe /Fe shifts the Mechanism of NO Reduction. The molecular mechanism of the NO Soret from 418 to 424 nm and the Q band from 530 to 546 nm reduction by NOR is still under debate. Two mechanisms have (Fig. 11). The FTIR of the solid samples from the reaction been proposed for binding and reduction of NO at the active site: Ϫ1 mixture reveals a band at 1,924 cm , which indicates the A“trans” mechanism invokes binding of two NO molecules to formation of a ferric heme nitrosyl species, and another weak Ϫ1 the heme-iron and nonheme iron separately at the fully reduced band at 1,812 cm , which is indicative of a ferrous nonheme active site, whereas a ‘‘cis’’ mechanism suggests binding of both nitrosyl species (Fig. S7). EPR data from the NO adduct with NO molecules to only one iron metal (typically Fe ). Recent LFeIII/FeII indicates a mixture of a heme Fe-NO (signal at g ϭ B ϭ quick-freezing EPR (15) indicates the formation of both heme- 2.0), a nonheme Fe-NO species (signal at g 4.0), and a iron nitrosyl and nonheme-iron nitrosyl species, supporting the high-spin FeIII species (at g ϭ 6) (Table 1). This demonstrates “trans” mechanism. Meanwhile, the mixed-valent state (heme- that the product of LFeIII/FeIIϩNO is a mixture of LFeIII-NO/ FeIII/Fe II) because of the low midpoint potential of heme b FeII-NO and LFeII-NO/FeIII. The ferric heme nitrosyl is EPR B 3 (heme b , E ϭ 60 mV; Fe , E ϭ 320 mV), was suggested to silent, but an N-O stretch is observed in the IR. This implies that 3 m B m be the active form of the enzyme (10). Therefore, it had been NO binding to the LFeIII/FeII complex leads to some electron proposed that the binding and reduction of NO occurs exclu- transfer from the ferrous nonheme to the ferric heme center, resulting in an equilibrium of three iron nitrosyl species: ferric sively on FeB, leaving the heme Fe uninvolved during the heme nitrosyl, ferrous heme nitrosyl, and ferrous nonheme nitrosyl. In any case, these mixed-valence species do not form N2O. Discussion The Synthetic Heme/Nonheme Diiron Compound Is a Functional NOR Model. In this study, a synthetic model compound is reported that integrates the essential features proposed for a bacterial NOR active site; namely, a heme active site with a covalently attached imidazole ligand and a nonheme site coordinated via three imidazole ligands. This model possesses the functional NO

†The reaction of LFeIII with NO led to the formation of LFeIII-NO, which is characterized by UV-vis, IR, and resonance Raman; no reduction of ferric heme to form heme Fe(II)-NO was Fig. 8. EPR data of the LFeII-NO (solid line), LZnII/FeII-NO (dotted line), and .observed. LFeII/FeII؉NO (dotted-to-dashed line) complexes

15662 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808606105 Collman et al. Downloaded by guest on September 29, 2021 Fig. 9. FTIR spectra of LFeII/FeII (solid line), LFeII/FeII ؉14NO (dotted line), and (LFeII/FeII ؉15NO (dashed line

catalytic process (10, 11). In addition, it has also been suggested that two molecules of NO bind consecutively at the heme Fe3ϩ site to form a hyponitrite intermediate, which is thought to Scheme 1. Reactions of mono- and bis-iron complexes with NO. (A) LFeII ϩ decay, giving the ferric heme, N2O, and H2O. A six-coordinate NO. (B) LZnII/FeII ϩ NO. (C) LFeIII/FeII ϩ NO. (D) LFeII/FeII ϩ NO. 3ϩ heme Fe -NO species of cytochrome cbb3 oxidase and a hyponitrite species in the Fe-Cu dinuclear center were detected by FTIR and the resonance Raman spectroscopy method, re- proximal imidazole is still coordinated to the heme iron. Such a spectively (34, 36). six-coordinate ferrous nitrosyl does not appear to be a ‘‘dead- We have shown that NO can readily bind to heme Fe and end’’ species as previously claimed (10). Instead, the bound nonheme Fe separately to form stable heme Fe-NO and non- nitrosyl can undergo N-N coupling with the adjacent nonheme heme Fe-NO species. This indicates that the enzymatic reaction nitrosyl to produce N2O. Praneeth et al. (37) demonstrated that probably proceeds by a “trans” mechanism. Two equivalents of a six-coordinate iron(II) porphyrin NO adduct with a proximal ⅐ NO react with the diiron center and form individual heme and imidazole ligand has a distinctive FeIINO character relative to a nonheme nitrosyl species. Then, the two adjacent bound ni- five-coordinate FeII-NO compound (37). Moreover, the en- trosyls undergo reductive coupling to form N2O (possibly hanced radical character of the heme nitrosyl should be advan- through a Fe-N(O)-N(O)-Fe intermediate), leaving both the tageous for the central (radical) N-N coupling step in the “trans” heme Fe and the nonheme Fe in an oxidized ferric state. It is not mechanism. On the other hand, a less reactive five-coordinate CHEMISTRY clear if a ␮-oxo diiron compound is formed as an intermediate FeII-NO would be a dead-end species, as demonstrated by a during this reaction, because excess NO may bind to the heme recent synthetic diiron model based on a five-coordinate heme Fe and cause the rupture of Fe-O-Fe bond, leading to the nitrosyl, which does not show any NO reductase activity (18). observed final LFeIII-NO/FeIII-OH compound. The reaction product of LFeII/FeII with two equivalents of NO did not show The Mixed-Valence Form of NOR Is Not Active for the Reduction of NO a ferric heme nitrosyl signal in the FTIR, and a small ion peak to N2O. It has been suggested that NO activation occurs with a ϩ at m/z 1,491 amu corresponding to a [LFe-O-Fe(MeCN)2] was mixed-valence form of the NO reductase with an oxidized heme b3 detected by electrospray mass spectroscopy. and a reduced nonheme FeB (10). Based on this, it was proposed It has been postulated that NO binding to the heme iron of that the binding of two molecules of NO happens exclusively on NOR or CcO causes bond cleavage between heme Fe and the either heme Fe or FeB, leaving the other metal essentially a witness. proximal imidazole producing a five-coordinate heme nitrosyl However, our studies demonstrated that reaction of a mixed- complex (5, 8). However, we show that binding NO to LFeII valence compound LFeIII/FeII with NO leads to a mixture of two III II II III results in a stable six-coordinate heme nitrosyl in which the species: LFe -NO/Fe -NO and LFe -NO/Fe .NoN2O was

Fig. 10. Resonance Raman of the reaction product of LFeII/FeII؉NO with Fig. 11. The optical spectra of LFeIII/FeII (solid line) and LFeIII/FeII ؉ NO 14NO (solid line) and 15NO (dotted line). (dotted line)

Collman et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 15663 Downloaded by guest on September 29, 2021 Table 1. Summary of spectroscopic features of LFeII-NO, LZnII/FeII-NO, LFeII/FeII؉NO, and LFeIII/FeIII-OMe ؉ NO Spectroscopy method LFeII-NO LZnII/FeII-NO LFeII/FeII؉NO LFeIII/FeIII؉NO LFeIII/FeII؉NO

IR 14NO/ 15NO (cmϪ1) 1,630/1,600 1,810/1,774 1,924/1,887 1,924/1,885 1,924, 1,812 (14NO) EPR S ϭ 1/2, g ϭ 2.08, S ϭ 3/2 g ϭ 4.0 S ϭ 1/2 g ϭ 2.07, 2.02, 1.96 S ϭ 1/2, g ϭ 6 after g ϭ 2.0 g ϭ 4.0 g ϭ 6 2.02, 1.97 reaction of NaOMe 14 Ϫ1 NAyϭ22 cm (1 eq): g ϭ 2.07, 15 Ϫ1 NAyϭ31 cm 2.00, 1.95

detected from either of these species, suggesting that a so-called glovebox, spotting on a KBr or NaCl palate, allowing the solvent to evaporate, ‘‘cis’’ mechanism is unlikely in the NO reduction by NOR. and then covering it by another palate and sealing the sides with parafilm. The palates containing the sample were sealed in a container and brought to the Conclusions IR spectrometer for measurement. Room-temperature UV-vis spectra were We have described a functional heme/nonheme nitric oxide recorded with a HP8452 diode array spectrophotometer. Mass spectra were II II obtained from the Stanford Mass Spectrometry Laboratory. The air-sensitive reductase model LFe /Fe that can reduce NO to N2O stoichio- sample solutions were prepared in a glovebox and sealed in gas-tight vials. metrically, leading to a bis-ferric product. NO binds to the heme They were brought to the spectrometer and injected into the instrument II Fe of LFe , producing a stable six-coordinate heme Fe-NO immediately before the measurement. II II complex, whereas binding of NO to a nonheme Fe of LZn /Fe EPR spectra were obtained by using a Bruker EMX spectrometer, ER 041 XG leads to a nonheme Fe-NO species. These results suggest that the microwave bridge, and ER 4102ST cavity. All X band samples were run at 77 K II II reaction of LFe /Fe with NO follows a “trans” mechanism: Two in a liquid nitrogen finger dewar. A Cu standard (1.0 mM CuSO4⅐5H2O with 2 molecules of NO bind to heme Fe and nonheme Fe separately, mM HCl and 2 M NaClO4) was used for spin quantitation of the EPR spectra. forming a heme Fe-NO and a nonheme Fe-NO species; then the Resonance Raman (rR) spectra were obtained by using a Princeton Instru- two adjacent nitrosyls undergo reductive coupling, producing ments ST-135 back-illuminated CCD detector on a Spex 1877 CP triple mono- chromator with 1,200, 1,800, and 2,400 grooves per millimeter holographic N2O and the di-ferric product. The mixed-valence form of our model compound LFeIII/FeII does not show any NO reduction spectrograph gratings. Excitation was provided by a Dye Laser (Stilbene 599; Coherent) that was energized by a Coherent Innova Sabre 25/7 Arϩ CW ion activity; instead, stable nitrosylated species were formed. Ex- laser. The laser line 425 nm (Ϸ10 mW) was used for excitation. The spectral periments focusing on the reaction intermediates to further resolution was Ͻ2cmϪ1. Sample concentrations were Ϸ1 mM in Fe. The clarify the reaction mechanism await completion. samples were either cooled to 77 K in a quartz liquid nitrogen finger dewar (Wilmad) and hand spun to minimize sample decomposition during scan Materials and Methods collection or cooled to 190–196 K by using a flow of liquid-N2-cooled He gas All reagents were obtained from commercial suppliers and used without in a spinner setup. further purification unless otherwise indicated. Fe(OTf)2(MeCN)2 was pre- In evaluating for the possibility of generation of N2O in the reaction pared according to literature procedures (38). Heme compound LFeII was mixture, the reaction of LFeII/FeII with purified NO was performed in dichlo- synthesized as reported (25). All air- and moisture-sensitive reactions were romethane, and a buffer solution was used to extract this organic layer and carried out under a nitrogen atmosphere in oven-dried glassware. Acetoni- then used for the activity assay. Simultaneously, PnN2OR enzyme was incu- trile, tetrahydrofuran, and dichloromethane were purified and dried by pass- bated in an excess of an anaerobic solution of methyl viologen and dithionite ing reagent-grade solvent through a series of two activated alumina columns in Tris buffer (pH Ϸ7.3), in the glovebox (required to activate the enzyme) (39). under nitrogen atmosphere. These solvents were further deoxygenated by Activity of the enzyme was determined spectrophotometrically, after the bubbling with nitrogen for 30 min in a nitrogen glove box. DMF was distilled oxidation of dithionite reduced methyl viologen at 600 nm by using a standard over molecular sieves and properly deoxygenated. Nitric oxide (NO) was protocol under anaerobic conditions (40, 41). The activity initiated by adding obtained from Matheson Gas Products or generated by adding saturated 20 ␮l of the 100-␮l buffer solution used to extract the reaction mixture in ϭ Ϫ1 Ϫ1 NaNO2 solution into a sulfuric acid solution (98% sulfuric acid /water 3:1). It CH2Cl2 was 43 ␮mol of N2O reduced min mg of N2OR. As a control, activity is purified by passage through a series of two thoroughly degassed 3.0 M KOH measured by initiating the reaction with NO-saturated buffer solution was 10 solutions and water. The saturated NO solution in acetonitrile or THF was units. A N2O concentration-vs.-activity curve shows that 43 units of activity made by bubbling purified NO gas through deoxygenated solvent in a gas- correspond to an N2O concentration of 1 mM. tight vial for 15 min. All reactions with NO (including making EPR and UV-vis samples) were carried out by injecting saturated NO solution into the sample ACKNOWLEDGMENTS. We thank Dr. Allis Chien of the Stanford University 15 solution in a glovebag purged and filled with nitrogen. The NO (99%) was Mass Spectrometry Group for mass spectrometry analysis. This material is purified by passing through a column packed with dry KOH powder under N2. based on work supported by National Institutes of Health Grant GM-69658 (to Infrared spectra were obtained on a Mattson Galaxy 4030 FT-IR spectrom- J.P.C.) and National Science Foundation Grant DMB0342807 (to E.I.S.). R.A.D. eter. Solid samples were prepared by dissolving a sample in solution in a is thankful for a Lavoisier Fellowship.

1. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol 8. Pinakoulaki E, Stavrakis S, Urbani A, Varotsis C (2002) Resonance Raman detection of Rev 61:533–616. a ferrous five-coordinate nitrosylheme b3 complex in cytochrome cbb3 oxidase from 2. Wasser IM, de Vries S, Moenne-Loccoz P, Schroder I, Karlin KD (2002) Nitric oxide in Pseudomonas stutzeri. J Am Chem Soc 124:9378–9379. biological denitrification: Fe/Cu metalloenzyme and metal complex NOx redox chem- 9. Pinakoulaki E, Varotsis C (2008) Resonance Raman spectroscopy of nitric oxide reduc- istry. Chem Rev 102:1201–1234. tase and cbb3 heme-copper oxidase. J Phys Chem B 112:1851–1857. 3. Hendriks J, et al. (1998) The active site of the bacterial nitric oxide reductase is a 10. Gronberg KLC, et al. (1999) A low-redox potential heme in the dinuclear center of dinuclear iron center. Biochemistry 37:13102–13109. bacterial nitric oxide reductase: Implications for the evolution of energy-conserving 4. Field SJ, et al. (2002) Spectral properties of bacterial nitric-oxide reductase— heme-copper oxidases. Biochemistry 38:13780–13786. Resolution of pH-dependent forms of the active site heme b3. J Biol Chem 11. Gronberg KLC, Watmough NJ, Thomson AJ, Richardson DJ, Field SJ (2004) Redox- 277:20146–20150. dependent open and closed forms of the active site of the bacterial respiratory nitric-oxide 5. Moenne-Loccoz P, de Vries S (1998) Structural characterization of the catalytic high- reductase revealed by cyanide binding studies. J Biol Chem 279:17120–17125. spin heme b of nitric oxide reductase: A resonance Raman study. J Am Chem Soc 12. Moenne-Loccoz P, et al. (2000) Nitric oxide reductase from Paracoccus denitrificans 120:5147–5152. contains an oxo-bridged heme/nonheme diiron center. J Am Chem Soc 122:9344–9345. 6. Pinakoulaki E, Ohta T, Soulimane T, Kitagawa T, Varotsis C (2005) Detection of the 13. Moenne-Loccoz P (2007) Spectroscopic characterization of heme iron-nitrosyl species and 2ϩ his-heme Fe -NO species in the reduction of NO to N2Obyba3-oxidase from Thermus their role in NO reductase mechanisms in diiron proteins. Nat Prod Rep 24:610–620. thermophilus. J Am Chem Soc 127:15161–15167. 14. Sakurai T, Nakashima S, Kataoka K, Seo D, Sakurai N (2005) Diverse NO reduction by 7. Pinakoulaki E, Varotsis C (2008) Nitric oxide activation and reduction by heme-copper Halomonas halodenitrificans nitric oxide reductase. Biochem Biophys Res Commun oxidoreductases and nitric oxide reductase. J Inorg Biochem 102:1277–1287. 333:483–487.

15664 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808606105 Collman et al. Downloaded by guest on September 29, 2021 15. Kumita H, et al. (2004) NO reduction by nitric-oxide reductase from denitrifying 28. Zhao Y, Brandish PE, Ballou DP, Marletta MA (1999) A molecular basis for nitric oxide bacterium Pseudomonas aeruginosa—Characterization of reaction intermediates that sensing by soluble guanylate cyclase. Proc Natl Acad Sci USA 96:14753–14758. appear in the single turnover cycle. J Biol Chem 279:55247–55254. 29. O’Keefe DH, Ebel RE, Peterson JA (1978) Studies of the oxygen binding site of cyto- 16. Collman JP, Yan YL, Lei JP, Dinolfo PH (2006) Efficient synthesis of trisimidazole and chrome P-450. Nitric oxide as a spin-label probe. J Biol Chem 253:3509–3516. glutaric acid bearing porphyrins: Ligands for active-site models of bacterial nitric oxide 30. Yonetani T, Yamamoto H, Erman JE, Leigh JS, Reed GH (1972) Electromagnetic prop- reductase. Org Lett 8:923–926. erties of hemoproteins. V. optical and electron paramagnetic resonance characteristics 17. Collman JP, Yan YL, Lei JP, Dinolfo PH (2006) Active-site models of bacterial nitric oxide of nitric oxide derivatives of metalloporphyrin-apohemoprotein complexes. J Biol reductase featuring tris-histidyl and glutamic acid mimics: Influence of a carboxylate Chem 247:2447–2455. ligand on FeB binding and the heme Fe/FeB redox potential. Inorg Chem 45:7581–7583. 31. Tomita T, Haruta N, Aki M, Kitagawa T, Ikeda-Saito M (2001) UV resonance Raman detection 18. Wasser IM, Huang HW, Moenne-Loccoz P, Karlin KD (2005) Heme/nonheme diiron(II) of a ligand vibration on ferric nitrosyl heme proteins. J Am Chem Soc 123:2666–2667. complexes and O2, CO, and NO adducts as reduced and substrate-bound models for the 32. Obayashi E, et al. (1997) Unique binding of nitric oxide to ferric nitric oxide reductase active site of bacterial nitric oxide reductase. J Am Chem Soc 127:3310–3320. from Fusarium oxysporum elucidated with infrared, resonance Raman, and X-ray 19. Ju TD, Woods AS, Cotter RJ, Moenne-Loccoz P, Karlin KD (2000) Dioxygen and nitric absorption spectroscopies. J Am Chem Soc 119:7807–7816. 5 II⅐⅐⅐ II ϩ oxide reactivity of a reduced heme/nonheme diiron(II) complex lex [( L)Fe Fe -Cl] . 33. Pinakoulaki E, Gemeinhardt S, Saraste M, Varotsis C (2002) Nitric-oxide reductase— Using a tethered tetraarylporphyrin for the development of an active site reactivity Structure and properties of the catalytic site from resonance Raman scattering. J Biol model for bacterial nitric oxide reductase. Inorg Chim Acta 297:362–372. Chem 277:23407–23413. 20. Wasser IM, et al. (2004) Synthesis and spectroscopy of ␮-oxo (O2-)-bridged heme/ 34. Stavrakis S, Pinakoulaki E, Urbani A, Varotsis C (2002) Fourier transform infrared evidence for nonheme diiron complexes: Models for the active site of nitric oxide reductase. Inorg a ferric six-coordinate nitrosylheme b3 complex of cytochrome cbb3 oxidase from Pseudomo- Chem 43:651–662. nas stutzeri at ambient temperature. J Phys Chem B 106:12860–12862. 21. Collman JP, Sunderland CJ, Berg KE, Vance MA, Solomon EI (2003) Spectroscopic 35. Miller LM, Pedraza AJ, Chance MR (1997) Identification of conformational substates evidence for a heme-superoxide/Cu(I) intermediate in a functional model of cyto- involved in nitric oxide binding to ferric and ferrous myoglobin through difference chrome c oxidase. J Am Chem Soc 125:6648–6649. Fourier transform infrared spectroscopy (FTIR). Biochemistry 36:12199–12207. 22. Collman JP, et al. (2007) A cytochrome c oxidase model catalyzes oxygen to water 36. Varotsis C, Ohta T, Kitagawa T, Soulimane T, Pinakoulaki E (2007) The structure of the reduction under rate-limiting electron flux. Science 315:1565–1568. hyponitrite species in a heme Fe-Cu binuclear center. Angew Chem Int Ed 46:2210–2214. 23. Boulatov R, Collman JP, Shiryaeva IM, Sunderland CJ (2002) Functional analogues of the dioxygen reduction site in cytochrome oxidase: Mechanistic aspects and possible 37. Praneeth VKK, Nather C, Peters G, Lehnert N (2006) Spectroscopic properties and electronic structure of five- and six-coordincate iron(II) porphyrin NO complexes: Effect effects of CuB. J Am Chem Soc 124:11923–11935. 24. Collman JP, Decreau RA, Yan YL, Yoon J, Solomon EI (2007) Intramolecular single- of the axial N-donor ligand. Inorg Chem 45:2795–2811. turnover reaction in a cytochrome c oxidase model bearing a Tyr244 mimic. J Am Chem 38. Hagen KS (2000) Iron(II) triflate salts as convenient substitutes for perchlorate salts: Crystal Soc 129:5794–5795. structures of [Fe(H2O)6](CF3SO3)2 and Fe(MeCN)4(CF3SO3)2. Inorg Chem 39:5867–5869. ␮ 25. Decreau RA, Collman JP, Yang Y, Yan YL, Devaraj NK (2007) Syntheses of hemoprotein 39. Ghosh S, et al. (2003) Activation of N2O reduction by the fully reduced 4-sulfide bridged models that can be covalently attached onto electrode surfaces by click chemistry. J Org tetranuclear Cu-Z cluster in nitrous oxide reductase. J Am Chem Soc 125:15708–15709. Chem 72:2794–2802. 40. Prudencio M, et al. (2000) Purification, characterization, and preliminary crystallo- 26. Andrew CR, George SJ, Lawson DM, Eady RR (2002) Six- to five-coordinate heme- graphic study of copper-containing nitrous oxide reductase from Pseudomonas nau- nitrosyl conversion in cytochrome cЈ and its relevance to guanylate cyclase. Biochem- tica 617. Biochemistry 39:3899–3907. istry 41:2353–2360. 41. Kristjansson JK, Hollocher TC (1980) First practical assay for soluble nitrous oxide 27. Makino R, et al. (1999) EPR characterization of axial bond in metal center of native and reductase of denitrifying bacteria and a partial kinetic characterization. J Biol Chem cobalt-substituted guanylate cyclase. J Biol Chem 274:7714–7723. 255:704–707. CHEMISTRY

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