Reversal of Cyanide Inhibition of Cytochrome c Oxidase by the Auxiliary Substrate Nitric Oxide
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m310359200
ISSN1083-351X
AutoresLinda L. Pearce, Emile L. Bominaar, Bruce C. Hill, Jim Peterson,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoNitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a3 by NO with reduction of the heme. The resulting nitrosylferroheme a3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only ∼50% in brain mitochondria. Nitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a3 by NO with reduction of the heme. The resulting nitrosylferroheme a3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only ∼50% in brain mitochondria. The reactions of cytochrome c oxidase with nitric oxide (NO) have recently been the subject of renewed scrutiny (1Torres J. Darley-Usmar V. Wilson M.T. Biochem. J. 1995; 312: 169-173Crossref PubMed Scopus (185) Google Scholar, 2Torres J. Sharpe M.A. Rosquist A. Cooper C.E. Wilson M.T. 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FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (526) Google Scholar, 14Tatoyan A. Giulivi C. J. Biol. Chem. 1998; 273: 11044-11048Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) raises a series of questions regarding the possible regulation of the electron transport chain by NO. Several catalytic cycles have been proposed (4Sarti P. Giuffre A. Forte E. Mastronicola D. Barone M.C. Brunori M. Biochem. Biophys. Res. Commun. 2000; 274: 183-187Crossref PubMed Scopus (133) Google Scholar, 6Sarti P. Giuffre A. Barone M.C. Forte E. Mastronicola D. Brunori M. Free Radic. Biol. Med. 2003; 34: 509-520Crossref PubMed Scopus (90) Google Scholar, 15Torres J. Cooper C.E. Sharpe M. Wilson M.T. J. Bioenerg. Biomembr. 1998; 30: 63-69Crossref PubMed Scopus (17) Google Scholar, 16Giuffre A. Barone M.C. Mastronicola D. D'Itri E. Sarti P. Brunori M. Biochemistry. 2000; 39: 15446-15453Crossref PubMed Scopus (68) Google Scholar, 17Pearce L.L. Kanai A.J. Birder L.A. Pitt B.R. Peterson J. J. Biol. Chem. 2002; 277: 13556-13562Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) to explain the consumption of NO by the oxidase while turning over in the presence of cytochrome c and oxygen. Some of these cycles implicitly treat the NO as an auxiliary substrate rather than simply a competitive inhibitor, to account for the observed production of nitrite in addition to the slowing of electron transfer rate. In some cases an oxyferryl intermediate of heme a3 is proposed and interaction of incoming NO with CuB assumed (15Torres J. Cooper C.E. Sharpe M. Wilson M.T. J. Bioenerg. Biomembr. 1998; 30: 63-69Crossref PubMed Scopus (17) Google Scholar, 16Giuffre A. Barone M.C. Mastronicola D. D'Itri E. Sarti P. Brunori M. Biochemistry. 2000; 39: 15446-15453Crossref PubMed Scopus (68) Google Scholar), whereas other schemes invoke the formation of a nitrosylheme a3 intermediate (4Sarti P. Giuffre A. Forte E. Mastronicola D. Barone M.C. Brunori M. Biochem. Biophys. Res. Commun. 2000; 274: 183-187Crossref PubMed Scopus (133) Google Scholar, 6Sarti P. Giuffre A. Barone M.C. Forte E. Mastronicola D. Brunori M. Free Radic. Biol. Med. 2003; 34: 509-520Crossref PubMed Scopus (90) Google Scholar, 17Pearce L.L. Kanai A.J. Birder L.A. Pitt B.R. Peterson J. J. Biol. Chem. 2002; 277: 13556-13562Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Consequently, clarification of which (if either) of the two centers of the binuclear pair, heme a3 or CuB, preferentially reacts with NO to form detectable derivatives should be of some value in assessing the relative likelihood of one proposed scheme over another as conditions are varied.When metalloenzymes have multiple binding sites for small molecular substrates and inhibitors, especially within the same active site domain, studies with mixed-ligand adducts can often reveal useful details of the coordination chemistry. Here we report the results of an investigation into the preferred site of NO reaction with bovine heart cytochrome c oxidase in the presence of the potent competitive inhibitor, cyanide, both under conditions of turnover and where the enzyme has been allowed to equilibrate in the presence of NO, cyanide, and reducing equivalents. The findings we report appear to be surprising in two respects. First, the inhibitory effects of NO plus cyanide toward the enzyme are not additive; NO actually eliminates cyanide inhibition. Second, contrary to the observations of earlier authors (18Stevens T.H. Bocian D.F. Chan S.I. FEBS Lett. 1979; 97: 314-316Crossref PubMed Scopus (24) Google Scholar, 19Stevens T.H. Brudvig G.W. Bocian D.F. Chan S.I. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3320-3324Crossref PubMed Scopus (101) Google Scholar, 20Brudvig G.W. Stevens T.H. Chan S.I. Biochemistry. 1980; 19: 5275-5285Crossref PubMed Scopus (215) Google Scholar, 21Stevens T.H. Chan S.I. J. Biol. Chem. 1981; 256: 1069-1071Abstract Full Text PDF PubMed Google Scholar), we find that cytochrome c oxidase displays a marked tendency to form NO adducts of heme a3 that are clearly 5-coordinate. The results have bearing on two related issues addressed under "Discussion." These are the likely mechanism of NO oxidation by the enzyme under conditions of high electron flux and the apparent existence of a cyanide-insensitive "pool" of cytochrome c oxidase in vivo.MATERIALS AND METHODSEnzymes and Reagents—Cytochrome c oxidase (EC 1.9.3.1, complex IV) was isolated from beef heart pericardium without the preparation of Keilin-Hartree particles (requiring acidification to pH < 6) common to most procedures. Using intact mitochondria, complex IV was separated from the other components of the electron transport chain by deoxycholate extraction as described by Ragan et al. (22Ragan C.I. Wilson M.T. Darley-Usmar V. Lowe P.N. Darley-Usmar V. Rickwood D. Wilson M.T. Mitochondria: A Practical Approach. IRL Press, Oxford1987: 79-112Google Scholar) and then finally purified by ammonium sulfate/cholate fractionation according to the procedure of Yonetani (23Yonetani T. Maehly A.C. Biochemical Preparations. Vol. 11. John Wiley & Sons, New York1966: 14-20Google Scholar). This method (in which the pH is maintained in the range of 7.8 to 8.0 throughout) yields good activity preparations of the oxidase that exhibit a 424 nm Soret absorption maximum, do not exhibit a g = 12 EPR signal, rapidly bind cyanide, and rapidly react with NO, which are characteristics of the pulsed form of the enzyme (24Giuffre A. Stubauer G. Brunori M. Sarti P. Torres J. Wilson M.T. J. Biol. Chem. 1998; 273: 32475-32478Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Moreover, the preparations usually persist in the pulsed form when stored overnight at 5–6 °C or for months (at least) in frozen (glassing) solution at 77 K. The enzyme was determined to be spectroscopically pure if the 444–424 nm ratio for the reduced enzyme was 2.2 or higher (25Gibson Q. Palmer G. Wharton D. J. Biol. Chem. 1965; 240: 915-920Abstract Full Text PDF PubMed Google Scholar). Derivatives were prepared in 50 mm potassium phosphate, 1 mm in sodium EDTA, and 0.1% in lauryl maltoside, pH 7.4–7.8, to concentrations of 10–80 μm (in enzyme). Enzyme concentrations were determined as total heme a using the differential (absorption) extinction coefficient of Δϵ604 = 12 mm-1 cm-1 for the reduced minus oxidized spectrum of the enzyme (26van Gelder B.F. Biochim. Biophys. Acta. 1966; 118: 36-46Crossref PubMed Google Scholar). Concentrations throughout are given on a per enzyme concentration basis (not per [heme a]). Ferrocytochrome c:O2 oxidoreductase activity was determined employing the high ionic strength method of Sinjorgo et al. (27Sinjorgo K.M. Durak I. Dekker H.L. Edel C.M. Hakvoort T.B. van Gelder B.F. Muijsers A.O. Biochim. Biophys. Acta. 1987; 893: 251-258Crossref PubMed Scopus (39) Google Scholar). Using this assay, we obtain a turnover number with respect to cytochrome c of 340 (±30) s-1 (0.1 m sodium phosphate, 0.1% lauryl maltoside, pH 7.4, 22 °C) similar to that of the bovine enzyme isolated from a variety of tissues by others (27Sinjorgo K.M. Durak I. Dekker H.L. Edel C.M. Hakvoort T.B. van Gelder B.F. Muijsers A.O. Biochim. Biophys. Acta. 1987; 893: 251-258Crossref PubMed Scopus (39) Google Scholar).A typical heme a extraction, maintaining nominally anaerobic conditions and under subdued light, was as follows. Approximately 2 ml of 0.26 mm (in total heme a) cytochrome c oxidase solution was taken, and 8 drops of concentrated sulfuric acid were added to precipitate the protein. After centrifugation (7000 × g, 22 °C, 2 min) the supernatant was discarded and heme a extracted from the pellet (22 °C, 20 min, frequent vortexing) into 1 ml of dimethylformamide. The concentration of heme a (80–100 μm) was determined using the extinction coefficient ϵ558 = 24 mm-1 cm-1 reported for the pyridine hemochrome (28Bartsch R.G. Methods Enzymol. 1971; 23: 344-363Crossref Scopus (197) Google Scholar). The procedure also results in the extraction of copper from the enzyme which appears in the final dimethylformamide solution as a stable Cu(II) species that is not reducible by ascorbic acid. EPR signals arising from this species have been removed from the nitrosylferroheme a spectra of Fig. 2, C and D, by subtracting the spectra of control samples prepared without adding NO.All reagents were ACS grade or better, used without further purification, and unless stated to the contrary, were purchased from Aldrich or Sigma. Sodium dithionite, 87% minimum assay (+H2O), was obtained from EM Science.13C sodium cyanide, 99%, was obtained from Cambridge Isotope Laboratories. Argon and nitric oxide gases were obtained from Matheson Incorporated. Nitric oxide was scrubbed with water and KOH pellets prior to use and added to enzyme samples volumetrically with gas-tight syringes. Buffered solutions never exhibited any significant change of pH (i.e. 20 subunits, rather than the 13 minimally required). The nitrosylferrocytochrome a3 (g = 2.09, 2.00, 1.97, and 9-line hyperfine structure on g = 2.00, ANO = 21 G, AHis = 7 G) resulting from the reduction of Hartzell-Beinert enzyme preparations in Tween 80 detergent incubated with NO have been investigated intensely (19Stevens T.H. Brudvig G.W. Bocian D.F. Chan S.I. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3320-3324Crossref PubMed Scopus (101) Google Scholar, 20Brudvig G.W. Stevens T.H. Chan S.I. Biochemistry. 1980; 19: 5275-5285Crossref PubMed Scopus (215) Google Scholar). Studies using15NO and the yeast enzyme from cells grown in15N-labeled histidine-rich media (18Stevens T.H. Bocian D.F. Chan S.I. FEBS Lett. 1979; 97: 314-316Crossref PubMed Scopus (24) Google Scholar, 21Stevens T.H. Chan S.I. J. Biol. Chem. 1981; 256: 1069-1071Abstract Full Text PDF PubMed Google Scholar) demonstrated conclusively that (i) the NO was bound to ferrocytochrome a3 and (ii) the EPR signal was further split by the presence of the proximal histidine ligand of the enzyme.The EPR spectrum obtained immediately following the addition of NO (1 atm) to a reduced sample of the current pulsed oxidase preparation is shown in Fig. 2A. Unlike the previously reported EPR spectrum of nitrosylferrocytochrome a3 derived from other preparations dispersed in Tween 80, the present data consist of overlapping spectra containing both 3-line (A = 18 G) and 9-line (A ∼ 20 G and A ∼ 7 G) hyperfine features. The two sets of signals clearly indicate a mixture of at least two products; one is the 6-coordinate species described above and the other (minority species) a new 5-coordinate derivative (see below).It has been shown repeatedly by magnetic circular dichroism spectroscopy (30Eglinton D.G. Johnson M.K. Thomson A.J. Gooding P.E. Greenwood C. Biochem. J. 1980; 191: 319-331Crossref PubMed Scopus (47) Google Scholar, 31Johnson M.K. Eglinton D.G. Gooding P.E. Greenwood C. Thomson A.J. Biochem. J. 1981; 193: 699-708Crossref PubMed Scopus (32) Google Scholar, 32Hill B.C. Brittain T. Eglinton D.G. Gadsby P.M. Greenwood C. Nicholls P. Peterson J. Thomson A.J. Woon T.C. Biochem. J. 1983; 215: 57-66Crossref PubMed Scopus (30) Google Scholar, 33Thomson A.J. Greenwood C. Gadsby P.M. Peterson J. Eglinton D.G. Hill B.C. Nicholls P. J. Inorg. Biochem. 1985; 23: 187-197Crossref PubMed Scopus (37) Google Scholar) that in the presence of excess reductant, cyanide-bound heme a3 is 6-coordinate and remains in the ferric form, whereas the other metal ion centers become reduced. Almost certainly, this partially reduced cyanide adduct is the major inhibited form in turnover experiments where cyanide is present. The EPR spectrum of the partially reduced cyanide adduct (10 mm in sodium cyanide plus excess sodium dithionite) is broad but readily detectable at 15 K (Fig. 2B, broken line). Treatment of this inhibited species with NO (1 atm) results in the quantitative formation of a new derivative exhibiting a characteristic EPR signal containing a distinct 3-line hyperfine pattern (A = 18 G) (Fig. 2B, solid line). This spectrum is predicted in the case of a single electron spin coupled to the three allowed orientations (MI = -1, 0, +1) of a nucleus like14N with nuclear spin IN = 1 and is sometimes referred to as "superhyperfine" structure if it is known that the electron and nuclear spins are not mainly localized on the same atom. We have also obtained qualitatively similar results starting from preparations of the dimeric resting enzyme dispersed in Tween 80 detergent (not shown); but in this case, the derivative exhibiting the 3-line hyperfine signals appeared to be only one of at least two species formed. The nitrosyl complexes of ferrohemes have been shown to have 5-coordinate structures in the absence of other strong-field ligands by elemental analysis of pure crystalline model compounds (34Scheidt W.R. Frisse M.E. J. Am. Chem. Soc. 1975; 97: 17-21Crossref PubMed Scopus (265) Google Scholar, 35Spaulding L.D. Chang C.C. Yu N.-T. Felton R.H. J. Am. Chem. Soc. 1975; 97: 2517-2525Crossref Scopus (273) Google Scholar, 36Yoshimura T. Bull. Chem. Soc. Jpn. 1978; 51: 1237-1238Crossref Google Scholar) and a structure determination by x-ray crystallography (34Scheidt W.R. Frisse M.E. J. Am. Chem. Soc. 1975; 97: 17-21Crossref PubMed Scopus (265) Google Scholar). Moreover, complexes of this type (NO-bound, d7, 5-coordinate hemes) have been demonstrated unequivocally to exhibit distinct 3-line hyperfine EPR spectra like those shown in Fig. 2 (37Wayland B.B. Olson L.W. J. Chem. Soc. Chem. Commun. 1973; : 897-898Crossref Google Scholar, 38Yoshimura T. Inorg. Chim. Acta. 1986; 125: L27-L29Crossref Scopus (9) Google Scholar). To better illustrate the point, the EPR spectrum of the nitrosyl adduct of extracted ferroheme a is shown in Fig. 2C (solid line), and also the spectrum of the analogous enzyme derivative has been superimposed (broken line) for comparison. It is to be stressed that the 5-coordinate nature of the complex is unambiguously demonstrated by the nitrosylferroheme a EPR spectrum of Fig. 2C. If two axial nitric oxide ligands were bound, they would either couple antiferromagnetically, resulting in no EPR signal, or couple ferromagnetically, leading to a more complicated signature than the observed 3-line hyperfine. The effect of introducing a sixth nitrogenous ligand (e.g. pyridine) on the EPR spectrum of the extracted nitrosylferroheme a EPR spectrum is shown in Fig. 2D. Clearly, the spectrum of the nitrosyl enzyme derivative formed in the presence of cyanide does not look at all like the 6-coordinate case.In summary, the EPR data convincingly demonstrate that substitution of cyanide by NO at heme a3 in the partially reduced cyanide adduct (starting with monomeric pulsed enzyme) renders the heme a3 site 5-coordinate (Fig. 2B). To verify that the enzyme spectrum of Fig. 2B (solid line) represents a species in which cyanide is no longer bound to heme a3 after NO addition, samples made with13C-labeled cyanide were examined and found to contain no additional hyperfine features (not shown), confirming the displacement of the cyanide ion. The overall similarity of the 3-line hyperfine features of Fig. 2, A and B, indicates that, contrary to the earlier findings of others, a fraction of the heme a3 can also be pentacoordinate in the fully reduced derivative prepared in the absence of cyanide. These results suggest that the histidine ligand proximal to heme a3 is more labile than previously thought, indicating some conformational flexibility at the binuclear pair. In support of this observation, it is noteworthy that the proximal ligand-to-heme a3 bond length in the crystal structure of the Thermus thermophilus enzyme is significantly longer than in other reported structures (39Soulimane T. Buse G. Bourenkov G.P. Bartunik H.D. Huber R. Than M.E. EMBO J. 2000; 19: 1766-1776Crossref PubMed Scopus (404) Google Scholar).Electronic Absorption Spectroscopy—In Fig. 3A the electronic absorption spectra of two NO adducts of cytochrome c oxidase are shown. The first is formed by the addition of NO to the dithionite-reduced enzyme (dashed line) and the second by the addition of NO to the partially reduced cyanide adduct (solid line). In the case of the second species, the order in which cyanide, NO, and reducing agent are added is not important, as the same derivative was always obtained (result confirmed by EPR spectroscopy; data not shown). These data appear to be in agreement with the EPR spectra of Fig. 2, A and B; that is, in the presence and absence of cyanide somewhat different NO adducts are obtained.Fig. 3Electronic absorption spectra of bovine cytochrome c oxidase at 22 °C, 1.00 cm path length.A, pulsed monomeric oxidase (8 μm in 0.1% lauryl maltoside, 25 mm phosphate, 1 mm EDTA, pH 7.4) reduced with sodium dithionite plus 1 atm of NO (dashed line) and after subsequent addition of 1 mm cyanide (solid line). B, Soret region of the enzyme (8 μm in 0.1% lauryl maltoside, 25 mm phosphate, 1 mm EDTA, pH 7.4): pulsed monomeric enzyme (solid line); resting monomeric enzyme (dashed line); with 1 mm sodium nitrite added to the pulsed monomeric enzyme (dotted line). C, pyridine hemochrome derived from extracted heme a (50 μl of buffered enzyme solution, 0.70 ml of 0.5 m NaOH, 0.25 ml of pyridine, excess solid sodium dithionite) demonstrating the absence of other contaminating hemes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because NO is converted to nitrite by cytochrome c oxidase (2Torres J. Sharpe M.A. Rosquist A. Cooper C.E. Wilson M.T. FEBS Lett. 2000; 475: 263-266Crossref PubMed Scopus (103) Google Scholar, 17Pearce L.L. Kanai A.J. Birder L.A. Pitt B.R. Peterson J. J. Biol. Chem. 2002; 277: 13556-13562Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) it was important to establish whether any of the species formed in the presence of excess NO were nitrite adducts. As shown in Fig. 3B, the Soret maximum shifts from 424 nm in the pulsed enzyme (solid trace) to 419 nm (dotted trace) following the addition of excess sodium nitrite. Note that neither of these is the same Soret maximum found associated with the monomeric resting enzyme (421 nm, dashed line). Furthermore, we did not observe the appearance of a g = 12 EPR signal (not shown) upon formation of the nitrite adduct. In fact, when nitrite is added to the pulsed enzyme, some reduction of nitrite to NO occurs, and a very small amount of nitrosylferrocytochrome a3 appears in the EPR spectrum. Superficially, the absorption spectrum of the nitrite adduct resembles that of the dimeric resting cytochrome c oxidase in Tween 80 (Soret maximum 417–418 nm, not shown), but these are clearly not the same species. More importantly, there is no evidence in the spectra of Fig. 3A, where the Soret features are at 431 and 442 nm, for a shoulder in the vicinity of 419 nm. Consequently, none of the species formed in the presence of excess NO seem to be simple nitrite adducts.It is important to bear in mind that the Soret maxima of cytochrome c oxidase derivatives are subject to distortion by the presence of heme contaminants. Specifically, complex III of the electron transport chain can be an especially difficult impurity to eliminate (40Baker G.M. Noguchi M. Palmer G. J. Biol. Chem. 1987; 262: 595-604Abstract Full Text PDF PubMed Google Scholar). The pyridine hemochrome spectrum (28Bartsch R.G. Methods Enzymol. 1971; 23: 344-363Crossref Scopus (197) Google Scholar) of heme a extracted from the current enzyme preparations is shown in Fig. 3C. The presence of the visible region band at 588 nm establishes that the heme a macrocycle was not derivatized during the extraction procedure. More importantly, the absence of any peaks at 557 and 550 nm confirms that the preparations contained no significant amounts of heme b or heme c, respectively (we estimate <3% of these relative to heme a). The Soret maxima we document herein have, of course, all been determined employing preparations essentially free of other heme-containing impurities.DISCUSSIONMechanism of NO Turnover—In conjunction with the findings of others, the present results have implications concerning the likely mechanism(s) of NO turnover by cytochrome c oxidase. Sarti et al. (
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