Interaction of with a functional model of cytochrome c oxidase

James P. Collman*, Abhishek Dey, Richard A. Decreau, Ying Yang, Ali Hosseini, Edward I. Solomon, and Todd A. Eberspacher

Department of Chemistry, Stanford University, Stanford, CA 94305

Contributed by James P. Collman, May 6, 2008 (sent for review April 24, 2008) Cytochrome c oxidase (CcO) is a multimetallic enzyme that carries quantitative probes (e.g., EPR spectroscopy) and proposed that in out the reduction of O2 to H2O and is essential to respiration, the presence of O2, the NO is catabolized to nitrite, analytically providing the energy that powers all aerobic organisms by gen- detected, in the active site of CcO. This mechanism invoked Ϫ erating heat and forming ATP. The -binding heme a3 should generation of superoxide (O2 )byoxidationofCuB by O2 near an be subject to fatal inhibition by chemicals that could compete with NO-bound heme a3 site, which then decays via a peroxynitrite O2 binding. Near the CcO active site is another enzyme, NO intermediate. Although this seems reasonable, it is hard to inves- synthase, which produces the gaseous hormone NO. NO can tigate this mechanism in a protein active site because of the strongly bind to heme a3, thus inhibiting respiration. However, this presence of several other chromophores. CO also has an affinity for II I Ϫ6 disaster does not occur. Using functional models for the CcO active the catalytically active reduced Fe /Cu form of CcO (Kd ϭ 10 M) site, we show how NO inhibition is avoided; in fact, it is found that and competes with O2 binding to this active site (17). A classic Ϫ NO can protect the respiratory enzyme from other inhibitors such inhibitor of CcO, CN binds tightly to the fully oxidized site (Kd ϭ Ϫ6 as cyanide, a classic poison. 10 M), resulting in the formation of a low-spin heme a3 center (18). This binding shifts the reduction potential (E°) of heme a3 to amyl nitrite ͉ cyanide poisoning ͉ synthetic functional model ͉ EPR ϩ150 mV (E° ϭϩ350 mV resting), which should jeopardize the reduction of the fully oxidized FeIII/CuII form to the catalytically II I ϭ n this article we answer an important question: why is the active active Fe /Cu form by the adjacent heme a center (E° 250–300 mV), which could also inhibit the enzyme turnover (19). The Isite of the respiratory enzyme cytochrome c oxidase (CcO) not Ϫ severely inhibited by the diffusing hormone NO, produced by a common antidote for CN poisoning is to administer organic neighboring NO synthase (NOS)? Moreover, we demonstrate nitrites [e.g., amyl nitrite (AmN)], thiosulfate, and plentiful oxygen. that NO should protect CcO from other inhibitors. In this article we use a functional synthetic model complex of CcO CcO is a multicomponent membrane protein that catalyzes the in its monometallic Fe-only form (1) or in its functional bimetallic Ϫ Fe/Cu form (2) to study the interaction of NO with these inhibitors 4e reduction of O2 to H2O in all eukaryotes (1). The energy released in this process is used to produce a proton gradient across and suggest a mechanism of recovery from each inhibitor (20–22). the membrane, which provides the driving force for ATP synthesis. Results and Discussion The active site of CcO contains a bimetallic center comprising an The mononitrosyl complexes 1-NO and 2-NO were prepared porphyrin heme a3 and a tris-histidine-coordinated Cu (CuB) Ϫ in the distal pocket (Fig. 1) (2, 3). During catalytic turnover, the before studying reactivity with O2 and/or O2 . These NO com- II I plexes were generated by addition of 1 eq of NO (Scheme 1) to Fe /Cu center reduces O2, and the resulting oxidized site is reduced via electron transfer from two electron donors, heme a and their respective precursors. Their absorption spectra show sig- Cu , regenerating the FeII/CuI site. CcO is strongly inhibited by nificant shifts in the absorption bands: 428 nm (Soret) and 536 A nm to 425 nm (Soret) and 548 nm, respectively (Fig. 2). The EPR high levels of nitric oxide (NO) (KI ϭ 0.27 ␮M), carbon monoxide Ϫ of both 1-NO and 2-NO (Fig. 3) is characteristic of low-spin (CO) (KI ϭ 0.32 ␮M), and cyanide (CN )(KI ϭ 0.2 ␮M) (4). NO six-coordinate S ϭ 1/2 iron-nitrosyl species with gz ϭ 2.078, gy ϭ is produced continuously in the mitochondrial membrane adjacent 14 2.015, and gx ϭ 1.97. The multiple N superhyperfine in the Ay to CcO by mitochondrial NOS (5, 6). Apart from car exhaust and Ϫ1 Ϫ1 Ϫ (N ϭ 6cm ,N ϭ 22 cm ) and its perturbation by cigarette smoke, CN is produced naturally in the human body as Imz NO isotopically enriched 15NO (N ϭ 6cmϪ1,N ϭ 31 cmϪ1) are a metabolic by-product of cyanogenic glycosides that are present in Imz NO similar to data previously reported for CcO’s active site and bitter almonds, apple seeds, raw yucca, and cassava roots (7). CO six-coordinate iron nitrosyl with imidazole axial [sup- is produced in cells as a by-product of heme degradation (8). Any porting information (SI) Fig. S1] (23, 24). Addition of excess O sudden increase in the flux of these inhibitors leads to a surge of 2 to 1-NO, even after prolonged exposure, does not result in any partially reduced oxygen species in the cell, which eventually leads change, neither in the UV-visible (UV-Vis) nor in the EPR to cell death (9). NO has a high equilibrium binding constant (K Ͻ d spectra (1-NO-O ; Figs. 2A and 3A). However, with 2-NO, 10Ϫ9 M) and a fast second-order kinetic rate constant (k ϭϷ108 2 on addition of O shifts of the absorption maxima to 429 and 539 nm MϪ1⅐sϪ1) for reduced ferrous porphyrin active sites, including the 2 (Fig. 2B), which is similar to that of the ferrous form. Simulta- reduced heme a site of CcO (10, 11). Because of a constant flux 3 neous EPR experiments show loss of the characteristic iron- of NO at the CcO active site ([O ]/[NO] ϭ 1,000 in mitochondria), 2 nitrosyl S ϭ 1/2 signal and development of another S ϭ 1/2 signal, there must be a pathway to avoid inhibition. Inhibition of CcO by ϩ which is characteristic of a type 2 Cu2 species (gʈ ϭ 2.40, gЌ ϭ NO has been reported to be reversible in the presence of O2; that is, after the initial inhibition by NO, the O2-reducing activity of CcO is slowly regained in the presence of O2 (11–13). From optical Author contributions: J.P.C., A.D., and T.A.E. designed research; A.D., R.A.D., Y.Y., and A.H. studies of the interaction of oxymyoglobin (Mb-O2) with NO-bound performed research; E.I.S. contributed new reagents/analytic tools; A.D. analyzed data; and CcO, Sarti et al. (14, 15) proposed a mechanism that entails A.D. wrote the paper. dissociation of bound NO from the heme a3 site. However, this The authors declare no conflict of interest. proposal presents a dilemma, because dissociation of NO from *To whom correspondence should be addressed. E-mail: [email protected]. Ϫ1 heme a3 has a first-order rate of koff ϭ 0.004 s , which is orders of This article contains supporting information online at www.pnas.org/cgi/content/full/ magnitude slower than the rate necessary for normal CcO turnover. 0804257105/DCSupplemental. Pearce et al. (16) have investigated the same reaction by using more © 2008 by The National Academy of Sciences of the USA

9892–9896 ͉ PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804257105 Downloaded by guest on September 23, 2021 Fig. 1. The CcO active site along with the electron-transfer heme a site and model complexes 1 and 2.

Ϫ Ϫ 2.082, Aʈ ϭ 122 cm 1, AЌ ϭ 16 cm 1; Fig. 3B). Quantification should rapidly isomerize in organic solvents). We propose that 2ϩ I Ϫ of these EPR signals against a Cu standard indicates that both in the presence of distal Cu dioxygen is reduced in situ to O2 , 2-NO and 2-NO-O2 have one paramagnetic center. On the other which reacts with the iron-nitrosyl species, transforming it into Ϫ Ϸ hand, addition of 1 eq of O2 to 1-NO shifts the absorption bands the ferrous heme active site [the potential of the Cu site is 0 Ϫ Ϫ from 425 nm, 548 nm to 428 nm, 538 nm (1-NO-S in Figs. 2A and mV (18), which can reduce O2 to O2 ( 100 mV) in presence of ϭ Ϫ 3A) and results in decay of the iron-nitrosyl S 1/2 EPR signal. excess O2]. This O2 -dependent destruction of the stable ferrous No other new EPR signals are observed in 1-NO-S and 2-NO-O2 nitrosyl species provides a plausible mechanism for the recovery even at4K(Fig. S2), which suggests the absence of any high-spin of CcO from NO inhibition in the presence of both O2 and an ferric species. Thus, both the UV-Vis and EPR data of 1-NO-S electron from the CuB site. This reaction probably proceeds via II and 2-NO-O2 are consistent with an Fe end product. a peroxynitrite intermediate, which forms by a reaction between These results, summarized in Scheme 1, suggest that an superoxide and the NO complex. The peroxynitrite readily iron-nitrosyl species lacking a distal CuI is inert toward oxygen isomerizes into nitrate in organic solvents (25) (under physio- but is reactive toward superoxide, forming a ferrous species (no logical conditions, this peroxynitrite would be reduced to nitrite intermediates were detected in this reaction even at Ϫ78°C, at the fully reduced active site of CcO). which is not unexpected, because peroxinitrite species, a very Adding CO to a solution of the unsubstituted ferrous-heme 1 strong candidate for the intermediate, is very unstable and leads to sharpening of the Soret and a small red shift of the band CHEMISTRY

Ϫ Scheme 1. Proposed reaction mechanism of an NO-inhibited CcO model (2-NO) with O2 and Fe-only NO-bound ferrous porphyrin (1-NO) with O2 . The superstructures are not included for clarity.

Collman et al. PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 9893 Downloaded by guest on September 23, 2021 Fig. 4. Absorption (A) and EPR (B) spectra of CO and CN derivatives of 1 and Fig. 2. Absorption spectrum of the NO derivatives of 1 (A) and 2 (B) and their 2 and their reactions with NO and AmN, respectively. Ϫ reactions with O2 and O2 .

equimolar NO (␭max ϭ 425 nm, 548 nm; Fig. 4A, green) is added at 536 nm, which is indicative of the ferrous carbonyl complex to a solution of 1-CN (Fig. 4A, blue) or 1-CO (Fig. 4A, red) (the Ϫ 1-CO (␭max ϭ 427 nm, 538 nm; Fig. 4A, red). Adding 10 eq of substitution of CO by NO is much slower than that of CN ). This CNϪ to 1 produces a red shift in both the Soret and the Q band, result is further supported by the EPR spectrum of 1-CN-NO, Ϫ ϭ suggesting formation of a CN complex 1-CN (␭max ϭ 433 nm, which shows the characteristic S 1/2 iron-nitrosyl signal 541 nm; Fig. 4A, blue). Both of these molecules are either Ϸ3,200–3,600 G (Fig. 3A) after adding NO to the diamagnetic produced in the human body or present in our diet or environ- 1-CN species (Fig. 4B, green). Parallel experiments were also ment, and they are known to be potent inhibitors of CcO. We performed with the active site of myoglobin. The absorption have observed that both CO and CNϪ are replaced when spectrum of Mb-CO has a Soret at 423 nm and Q bands at 544 and 582 nm that are shifted to 424, 551, and 582 nm, respectively, after adding NO, indicating displacement of CO to form the NO complex (26, 27). AmN is commonly used as an immediate treatment for acciden- tal exposure to CN. When AmN is added to 2-CN (prepared by adding 10 eq of CNϪ to 2), an EPR spectrum is obtained (2-CN- AmN; Fig. 4B, red), which suggests a mixture of an oxidized Cu2ϩ (indicated by the four-line hyperfine) and S ϭ 1/2 iron-nitrosyl species (indicated by the feature at 3,400 G). The total spin integration against a Cu2ϩ standard shows the presence of two paramagnetic species in 2-CN-AmN. In contrast, no reaction was observed between AmN and copper free 1. We propose that AmN

Fig. 3. EPR spectra of the NO derivatives of 1 (A) and 2 (B) and their reactions Ϫ ϫ 3 Ϫ with O2 and O2 . Frequency, 9.38 MHz; power, 10 mW; gain, 5 10 ; modu- Fig. 5. UV-Vis spectrum of CN derivative of 1-OX and its subsequent Ϫ lation amplitude, 10 G; temperature, 77 K. reaction with O2 .

9894 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804257105 Collman et al. Downloaded by guest on September 23, 2021 Scheme 2. Proposed mechanism for recovery from inhibition via in situ NO generation from AmN. The ligand superstructure is not included for clarity.

is reduced to amyl alkoxide and NO by the reduced CuB, which In summary our results strongly support the hypothesis by Pearce becomes oxidized to Cu2ϩ (Scheme 2). This NO generated in situ et al. (16) regarding the mechanism for recovery from inhibition of Ϫ from AmN should replace the CN , forming an iron-nitrosyl CcO by NO. NO reacts with our heme a3 model to form an species at the heme a3 site. This hypothesis was also evaluated by iron-nitrosyl complex that is stable in O2. However, in the presence Ϫ adding AmN to 2-CO (Fig. S3). of a reduced CuB in the distal site, O2 generates O2 , which reacts CNϪ is known to bind the oxidized FeIII form with a much with the iron-nitrosyl complex, possibly via a peroxynitrite inter- II Ϫ higher affinity than to Fe . In CcO, CN binding shifts the mediate, regenerating a ferrous heme a3 site ready for turnover. reduction potential of the heme a3 site by Ϫ200 mV (i.e., from Given the high binding constant of NO with heme a3 and generation ϩ350 to ϩ150 mV against a normal electrode). At this of NO by NOS adjacent to the mitochondria, we propose that this potential the heme a3 site could not be reduced by heme a, which reaction recurs repeatedly during the normal turnover of CcO and has a reduction potential of only ϩ250 mV and is unchanged by plays a regulatory role in the functioning of CcO. NO also may be Ϫ CN binding to heme a3 (19). This Ϫ200 mV decrease in an active component in the defense of CcO against inhibitors such Ϫ reduction potential of heme a3 should inhibit turnover of CcO. as CN and CO. Because of its extremely high binding affinity for Ϫ Binding 1 eq of CN to the oxidized model 1-OX (␭max ϭ 415 nm, reduced heme a3, NO can replace either of these ligands, forming 519 nm; Fig. 5) in dichloromethane red shifts the UV-Vis a stable ferrous nitrosyl complex, which could then be oxidized by Ϫ Ϫ (1-OX-CN, ␭max ϭ 423 nm, 555 nm; Fig. 5). Although this CN O generated in situ, thereby regenerating the active enzyme. This 2 Ϫ complex is stable in O2, it is readily reduced to the ferrous form in situ generation of O should provide also a defense against CHEMISTRY Ϫ 2 nm, 538 nm; inhibitors that target the oxidized heme a3 site (anionic ligands such 428 ؍ by addition of 1 eq of O (1-OX-CN-S, ␭max 2 Ϫ Fig. 5 red). Scheme 3 represents a likely mechanism that as CNϪ,N , etc.) by reducing the oxidized site to the corresponding Ϫ 3 accounts for these events. In CcO the O2 could be produced in ferrous forms, which have much higher NO affinities. Indeed, an I Ϫ situ after the 1-electron reduction of O2 by the distal Cu as increase of O2 concentration has been observed in CcO during suggested by the reaction of 2-NO with O2 (see above). turnover when the heme a3 site is inhibited by ligand binding

Scheme 3. Proposed mechanism for recovery from CNϪ inhibition of ferric CcO. The ligand superstructure is not included for clarity.

Collman et al. PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 9895 Downloaded by guest on September 23, 2021 (28). Thus, a symbiotic interaction between a potent inhibitor The CO samples were prepared by the addition to CO gas to the reduced Ϫ complexes in a cuvette or EPR tube. The formation of the CO complexes was (NO) and a reactive oxygen species by-product (O2 ) should protect CcO from external inhibitors. While NO defends the confirmed by UV-Vis spectroscopy. NO (soln) or AmN was then added to them. Ϫ CNϪ complexes were prepared by adding N,N,N,N-tetrabutylammonium cya- reduced site, O2 defends the oxidized site. nide to reduced or oxidized samples. The formation of the CN complexes was Materials and Methods confirmed by UV-Vis and IR spectroscopy. NO (soln) or AmN was then added to them. All chemical manipulations were carried out under N atmosphere unless men- 2 EPR spectra were obtained by using a Bruker EMX spectrometer, ER 041 XG tioned otherwise. The synthesis of 1 and 2 has been reported previously (29). microwave bridge, and ER 4102ST cavity. All X-band EPR samples were run at These are generally synthesized as the FeII and FeII/CuI forms, which are oxidized 77 K in a liquid nitrogen finger Dewar. A Cu standard (1.0 mM CuSO ⅐5H O with stoichiometric amounts of ferrocenium tetrafluoroborate as and when 4 2 with 2 mM HCl and 2 M NaClO4) was used for spin quantitation of the EPR mentioned. KO2 and [18]crown6 were purchased from Aldrich Chemicals and used without further purification. High-purity NO was purchased from Praxair spectra. EPR data of the ferrous end product was obtained at liquid He temperature (4 K). and was further purified by bubbling it twice through 4 M KOH and H2O under N2 atmosphere. The solubility of NO in acetonitrile was taken to be 14 mM as The UV-Vis spectra were collected by using a Hewlett–Packard apparatus reported (30). AmN was purchased from Aldrich and passed through a column of 8452 glass cuvette (3-ml capacity) sealed with a 14/24 septum (solvent: dim- basic alumina before used to get rid of any amyl and nitrogen dioxide ethylformamide/dichloromethane; concentration in porphyrin: 1 ϫ 10Ϫ5 M). impurity that may have been present. For the NO ϩ O2 reactions, 1 eq of NO was added, and the formation of the ACKNOWLEDGMENTS. We thank Somdatta Ghosh for help with the acquisi- nitrosyl species was confirmed by UV-Vis and EPR spectroscopy. To the same tion of low-temperature EPR data. This work was funded by National Insti- sample, excess O2 gas or 1 eq of superoxide was added and monitored by tutes of Health Grant 5R01 GM-17880-35. R.A.D. is thankful for a Lavoisier UV-Vis and EPR spectroscopy. Fellowship.

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