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• W2068334315 abstract "The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa3) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a3reduction, which is extremely slow in the K354M mutant (k 1 = 0.09 ± 0.01 s−1;k 2 = 0.005 ± 0.001 s−1) but much faster in the D124N aa 3(k 1 = 21 ± 6 s−1;k 2 = 2.2 ± 0.5 s−1). NO causes a very large increase (>100-fold) in the rate constant of heme a3 reduction in the K354M mutant but only a ∼5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O2 when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a3 reduction is ∼100-fold faster than the extremely low turnover rate, we conclude that, contrary to O2, NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H+-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site. The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa3) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a3reduction, which is extremely slow in the K354M mutant (k 1 = 0.09 ± 0.01 s−1;k 2 = 0.005 ± 0.001 s−1) but much faster in the D124N aa 3(k 1 = 21 ± 6 s−1;k 2 = 2.2 ± 0.5 s−1). NO causes a very large increase (>100-fold) in the rate constant of heme a3 reduction in the K354M mutant but only a ∼5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O2 when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a3 reduction is ∼100-fold faster than the extremely low turnover rate, we conclude that, contrary to O2, NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H+-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site. cytochromec oxidase singular value decomposition enzyme with oxidized heme a3-CuB site enzyme with a single-electron reduced heme a3-CuB enzyme with a two-electron reduced heme a3-CuB. Cytochrome c oxidase (CcOX)1 contains a bimetallic active site (heme a3-CuB) where O2is reduced to H2O. This exergonic reaction is coupled to an active translocation of protons, generating a proton-motive force used for ATP synthesis (see Refs. 1Michel H. Biochemistry. 1999; 38: 15129-15140Crossref PubMed Scopus (241) Google Scholar and 2Zaslavsky D. Gennis R.B. Biochim. Biophys. Acta. 2000; 1458: 164-179Crossref PubMed Scopus (159) Google Scholar for reviews). Complete reduction of the heme a3-CuB center, a prerequisite for the reaction with O2, occurs via intramolecular electron transfer from heme a, which in turn is reduced by CuA, the metal center accepting electrons from cytochrome c. Protons (both scalar and vectorial) are made available in situ via two putative H+-conducting pathways, identified in the crystallographic structure (3Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1967) Google Scholar, 4Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1898) Google Scholar). These pathways, called K and D from the residues Lys-354 2The amino acid numbering is based on theP. denitrificans cytochrome c oxidase sequence.2The amino acid numbering is based on theP. denitrificans cytochrome c oxidase sequence. and Asp-124 of subunit I, play different roles in the mechanism, as extensively investigated by site-directed mutagenesis (see Refs. 1Michel H. Biochemistry. 1999; 38: 15129-15140Crossref PubMed Scopus (241) Google Scholar, 2Zaslavsky D. Gennis R.B. Biochim. Biophys. Acta. 2000; 1458: 164-179Crossref PubMed Scopus (159) Google Scholar, and 5Pfitzner U. Odenwald A. Ostermann T. Weingard L. Ludwig B. Richter O.M. J. Bioenerg. Biomembr. 1998; 30: 89-97Crossref PubMed Scopus (115) Google Scholar for reviews).The catalytic cycle of cytochrome c oxidase can be divided into a reductive and an oxidative part. In the reductive part, two electrons are sequentially transferred to the fully oxidized heme a3-CuB center called O, yielding the two-electron reduced site R via a single-electron reduced intermediate E. In the oxidative part, upon reaction with O2, R restores the fully oxidized enzymeO, by populating the O2 intermediatesP and F (depending on the redox state of heme a, two different P intermediates are formed, calledPM andPR ). The idea that theO → R process is the rate-determining step in the overall catalytic cycle is gaining further support (6Malatesta F. Sarti P. Antonini G. Vallone B. Brunori M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7410-7413Crossref PubMed Scopus (46) Google Scholar, 7Verkhovsky M.I. Morgan J.E. Wikström M. Biochemistry. 1995; 34: 7483-7491Crossref PubMed Scopus (117) Google Scholar, 8Brunori M. Giuffrè A. D'Itri E. Sarti P. J. Biol. Chem. 1997; 272: 19870-19874Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).Mutation of Lys-354 to M within the K pathway yields a virtually inactive enzyme, as shown for the Rhodobacter sphaeroides aa 3 (9Hosler J.P. Shapleigh J.P. Mitchell D.M. Kim Y. Pressler M.A. Georgiou C. Babcock G.T. Alben J.O. Ferguson-Miller S. Gennis R.B. Biochemistry. 1996; 35: 10776-10783Crossref PubMed Scopus (89) Google Scholar, 10Konstantinov A.A. Siletsky S. Mitchell D. Kaulen A. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9085-9090Crossref PubMed Scopus (324) Google Scholar, 11Jünemann S. Meunier B. Gennis R.B. Rich P.R. Biochemistry. 1997; 36: 14456-14464Crossref PubMed Scopus (94) Google Scholar), the Escherichia coli bo 3 (12Svennson M. Hallén S. Thomas J.W. Lemieux L.J. Gennis R.B. Nilsson T. Biochemistry. 1995; 34: 5252-5258Crossref PubMed Scopus (30) Google Scholar), and the Paracoccus denitrificans aa 3 (5Pfitzner U. Odenwald A. Ostermann T. Weingard L. Ludwig B. Richter O.M. J. Bioenerg. Biomembr. 1998; 30: 89-97Crossref PubMed Scopus (115) Google Scholar). This mutation affects primarily, but not exclusively (see Ref. 13Brändén M. Sigurdson H. Namslauer A. Gennis R.B. Ädelroth P. Brzezinski P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5013-5018Crossref PubMed Scopus (136) Google Scholar), the reductive part of the catalytic cycle (9Hosler J.P. Shapleigh J.P. Mitchell D.M. Kim Y. Pressler M.A. Georgiou C. Babcock G.T. Alben J.O. Ferguson-Miller S. Gennis R.B. Biochemistry. 1996; 35: 10776-10783Crossref PubMed Scopus (89) Google Scholar, 11Jünemann S. Meunier B. Gennis R.B. Rich P.R. Biochemistry. 1997; 36: 14456-14464Crossref PubMed Scopus (94) Google Scholar, 14Zaslavsky D. Gennis R.B. Biochemistry. 1998; 37: 3062-3067Crossref PubMed Scopus (45) Google Scholar); in the absence of O2 and with a large excess of reductant, heme a3 is reduced at an extremely low rate (time scale of several minutes) as compared with the wild type (time scale of tens of milliseconds). This reduction block is presumably due to an impaired H+ transfer in the K354M mutant, consistent with the loss of the millisecond phase in laser-triggered reverse electron transfer experiments observed with the analogous mutant of the R. sphaeroides enzyme (15Ädelroth P. Gennis R.B. Brzezinski P. Biochemistry. 1998; 37: 2470-2476Crossref PubMed Scopus (128) Google Scholar). Recently, two groups (16Ruitenberg M. Kannt A. Bamberg E. Ludwig B. Michel H. Fendler K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4632-4636Crossref PubMed Scopus (88) Google Scholar, 17Wikström M. Jasaitis A. Backgren C. Puustinen A. Verkhovsky M.I. Biochim. Biophys. Acta. 2000; 1459: 514-520Crossref PubMed Scopus (133) Google Scholar, 18Verkhovsky M.I. Tuukkanen A. Backgren C. Puustinen A. Wikström M. Biochemistry. 2001; 40: 7077-7083Crossref PubMed Scopus (47) Google Scholar) reported time-resolved electrometric measurements on liposome-reconstituted mutants of the P. denitrificans CcOX by laser excitation of ruthenium(II) bispyridyl. According to Ruitenberg et al. (16Ruitenberg M. Kannt A. Bamberg E. Ludwig B. Michel H. Fendler K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4632-4636Crossref PubMed Scopus (88) Google Scholar), injection of a single electron into the oxidized enzyme is coupled to an H+ transfer through the K pathway, linked to reduction of heme a. In contrast, Verkhovsky et al. (18Verkhovsky M.I. Tuukkanen A. Backgren C. Puustinen A. Wikström M. Biochemistry. 2001; 40: 7077-7083Crossref PubMed Scopus (47) Google Scholar) proposed that an H+ uptake through the K pathway controls the single-electron reduction of heme a3-CuB(O → E), impaired in the K354M mutant, whereas the formation of the two-electron reduced active site (E →R) would be coupled to an H+ uptake through the D pathway, as deduced from data on the inactive D124N mutant (17Wikström M. Jasaitis A. Backgren C. Puustinen A. Verkhovsky M.I. Biochim. Biophys. Acta. 2000; 1459: 514-520Crossref PubMed Scopus (133) Google Scholar). The first of the two protons taken upon reduction of the active site has been proposed to charge-compensate the reduction of CuB in the single-electron reduced active site via protonation of a putative OH− bound to this metal in the oxidized state (1Michel H. Biochemistry. 1999; 38: 15129-15140Crossref PubMed Scopus (241) Google Scholar, 17Wikström M. Jasaitis A. Backgren C. Puustinen A. Verkhovsky M.I. Biochim. Biophys. Acta. 2000; 1459: 514-520Crossref PubMed Scopus (133) Google Scholar).The effect of the K354M mutation is drastic in the reductive part of the catalytic cycle but much smaller in the oxidative part. As assessed by the flow-flash technique using the R. sphaeroides CcOX analogous to the K354M mutant, the fully reduced enzyme exposed to O2 becomes fully oxidized within ∼5 ms (15Ädelroth P. Gennis R.B. Brzezinski P. Biochemistry. 1998; 37: 2470-2476Crossref PubMed Scopus (128) Google Scholar), although without the formation of PR (13Brändén M. Sigurdson H. Namslauer A. Gennis R.B. Ädelroth P. Brzezinski P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5013-5018Crossref PubMed Scopus (136) Google Scholar). The loss of oxidase activity associated to the K354M mutation has been therefore assigned to the extremely slow formation of R (9, 11, 14), which is a prerequisite for the reaction with O2.Differently from O2, NO has been suggested to bind not only to R (19) but also to a single-electron reduced intermediateE (20, 21). This hypothesis, raised to account for the very low apparent K i for NO inhibition, although consistent with computer simulations (20Torres J. Darley-Usmar V.M. Wilson M.T. Biochem. J. 1995; 312: 169-173Crossref PubMed Scopus (185) Google Scholar, 21Giuffrè A. Sarti P. D'Itri E. Buse G. Soulimane T. Brunori M. J. Biol. Chem. 1996; 271: 33404-33408Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), is not yet supported by direct experimental evidence. In this report, we provide evidence for the reaction of NO with E by studying the kinetics of reduction of the K354M and D124N mutants of P. denitrificansCcOX in the presence of NO.EXPERIMENTAL PROCEDURESDodecyl-β-d-maltoside was purchased from Biomol(Hamburg, Germany); ascorbate, glucose oxidase, and catalase were purchased from Sigma; and ruthenium(III) hexamine was purchased from Aldrich. Stock solutions of NO (Air Liquide, Paris, France) were prepared by equilibrating degassed water with the pure gas at 1 atm ([NO] = 2 mm at 20 °C).The K354M and D124N mutants of cytochrome c oxidase fromP. denitrificans were purified according to Ref. 22Hendler R.W. Pardhasaradhi K. Reynafarje B. Ludwig B. Biophys. J. 1991; 60: 415-423Abstract Full Text PDF PubMed Scopus (92) Google Scholar and stored at −80 °C. Before use, the enzymes were equilibrated by dialysis (at 4 °C for at least 5 h) with the buffer used in the experiments (100 mm K+/phosphate, pH 7.0, + 0.1% dodecyl maltoside or 35 mm K+/phosphate, pH 7.0, + 50 mm KCl + 0.1% dodecyl maltoside). Cytochrome oxidase concentration is expressed in terms of functional units (aa 3) using the extinction coefficient Δεred-ox, 444 = 156 mm−1cm−1.Stopped-flow experiments were carried out with a DX.17MV Applied Photophysics instrument equipped with a diode array (Leatherhead, UK). The mixing apparatus allows rapid mixing of equal volumes of solutions either in a simple or a sequential mode; in the latter mode, two solutions are premixed, and after a preset delay, they are mixed again with another solution. The instrument has a 1-cm light path and can acquire absorption spectra with an acquisition time of 2.5 ms. In a typical experiment, ascorbate (80 mm) and ruthenium hexamine (4 mm) are premixed with N2-equilibrated buffer (with or without NO), and the resulting solution is mixed after a 100-ms delay with degassed oxidized CcOX at 20 °C. This protocol prevents prolonged incubation of reductants with NO. Contaminant oxygen was scavenged with glucose and catalytic amounts of glucose oxidase and catalase. After the second mixing, absorption spectra were collected as a function of time according to a logarithmic scale. Data analysis was carried out using the software MATLAB (The MathWorks, South Natick, MA). Spectral smoothing and deconvolution were performed by using the singular value decomposition (SVD) algorithm according to Henry and Hofrichter (23Henry E.R. Hofrichter J. Methods Enzymol. 1992; 210: 129-192Crossref Scopus (599) Google Scholar) or by the pseudoinverse algorithm.The stoichiometry of NO binding to the K354M CcOX has been measured according to Stubauer et al. (24Stubauer G. Giuffrè A. Brunori M. Sarti P. Biochem. Biophys. Res. Commun. 1998; 245: 459-465Crossref PubMed Scopus (67) Google Scholar) by using a NO-selective Clark-type electrode (ISO-NO, World Precision Instruments). The electrode is calibrated using aliquots of NO-saturated water added to the degassed buffer and, after the addition of CcOX, the concentration of NO in solution is monitored.RESULTSIn the present investigation, we studied by stopped-flow spectrophotometry the kinetics of reduction of the K354M and the D124N mutants of the P. denitrificans CcOX both in the presence and in the absence of NO. As shown in Fig.1, the K354M mutation yields a dramatic decrease in the rate of heme a3 reduction, consistent with the literature (9Hosler J.P. Shapleigh J.P. Mitchell D.M. Kim Y. Pressler M.A. Georgiou C. Babcock G.T. Alben J.O. Ferguson-Miller S. Gennis R.B. Biochemistry. 1996; 35: 10776-10783Crossref PubMed Scopus (89) Google Scholar, 11Jünemann S. Meunier B. Gennis R.B. Rich P.R. Biochemistry. 1997; 36: 14456-14464Crossref PubMed Scopus (94) Google Scholar, 14Zaslavsky D. Gennis R.B. Biochemistry. 1998; 37: 3062-3067Crossref PubMed Scopus (45) Google Scholar, 17Wikström M. Jasaitis A. Backgren C. Puustinen A. Verkhovsky M.I. Biochim. Biophys. Acta. 2000; 1459: 514-520Crossref PubMed Scopus (133) Google Scholar). Upon mixing anaerobically oxidized K354M CcOX with a large excess of ascorbate and ruthenium hexamine, heme a reduction is very fast (∼7 ms), whereas reduction of heme a3 is extremely slow (>500 s, Fig. 1 A). SVD analysis of the latter process shows a single significant optical component (corresponding to the reduced minus oxidized heme a3 spectrum), displaying a biphasic time course (Fig.1 C). Best fit of the time course yieldsk 1 = 0.09 ± 0.01 s−1 andk 2 = 0.005 ± 0.001 s−1 for the two phases, accounting for ∼30 and 70% of the total amplitude, respectively. We cannot exclude that the intrinsic reduction rate might be even slower than observed given that over the very long time scale explored (500 s), the high intensity light beam of the diode array instrument causes partial enzyme reduction even in the absence of reductants (data not shown). In agreement with others (5Pfitzner U. Odenwald A. Ostermann T. Weingard L. Ludwig B. Richter O.M. J. Bioenerg. Biomembr. 1998; 30: 89-97Crossref PubMed Scopus (115) Google Scholar, 9Hosler J.P. Shapleigh J.P. Mitchell D.M. Kim Y. Pressler M.A. Georgiou C. Babcock G.T. Alben J.O. Ferguson-Miller S. Gennis R.B. Biochemistry. 1996; 35: 10776-10783Crossref PubMed Scopus (89) Google Scholar, 11Jünemann S. Meunier B. Gennis R.B. Rich P.R. Biochemistry. 1997; 36: 14456-14464Crossref PubMed Scopus (94) Google Scholar), we conclude that the very slow heme a3 reduction may account for the extremely slow turnover observed with O2(∼0.02 mol of O2/mol of CcOX × s at 170 μm cytochrome c)A different scenario is observed when the reduction of the K354M CcOX is carried out in the presence of NO (Fig. 1 B). In these experiments, the stopped-flow apparatus was used in the sequential mixing mode to prevent the prolonged incubation of NO with the reductants in the stopped-flow syringe to avoid NO loss (see “Experimental Procedures”). At 500 μm NO (concentration after mixing), heme a reduction is again complete within a few milliseconds. In this case, however, the end point species (i.e. the fully reduced enzyme with NO bound to heme a3) is already populated after about 10 s, indicating a much faster internal electron transfer in the presence of NO. SVD analysis of the absorption spectra collected from 7 ms up to 10 s reveals only a single optical component corresponding to the [heme a32+-NO]-[heme a33+] difference spectrum, indicating that NO binding is rate-limited by (and thus, apparently synchronous with) heme a3 reduction. Analysis of the time course in Fig. 1 C shows that the time course of heme a32+-NO formation is biphasic with rate constants of k 1 = ∼8.9 s−1 andk 2 = ∼0.6 s−1, the two phases having similar amplitude. Thus, NO seems not to interfere with heme a reduction (very fast both with and without NO) but clearly drives heme a3 reduction, which occurs in the presence of NO at least 100-fold faster than in its absence (Fig. 1 C).The experiments reported above were extended to the D124N mutant (Fig. 2). In the latter mutant, similarly to the K354M mutant, the reduction of heme a is fast both in the presence and in the absence of NO, being complete within a few milliseconds after mixing with reductant (Fig. 2, A andB). In agreement with the literature (17Wikström M. Jasaitis A. Backgren C. Puustinen A. Verkhovsky M.I. Biochim. Biophys. Acta. 2000; 1459: 514-520Crossref PubMed Scopus (133) Google Scholar), the enzyme is completely reduced within a few seconds even in the absence of NO (compare Figs. 1 A and 2 A), and thus, heme a3 reduction is much faster than in the case of the K354M mutant. Also, for the D124N mutant, heme a3 reduction is biphasic with k 1 = 21 ± 6 s−1and k 2 = 2.2 ± 0.5 s−1(relative amplitudes ∼70 and 30%, respectively). Complete reduction of the D124N mutant is accelerated in the presence of NO (Fig.2 B) and, for instance, at 500 μm NO, the formation of the heme a32+-NO complex proceeds at k 1 = 74 ± 11 s−1and k 2 = 3.6 ± 1.6 s−1(relative amplitudes ∼60 and 40%, respectively). Thus, we conclude that in both mutants, the addition of NO increases the rate of internal electron transfer; however, this increase corresponds to a factor of ∼5 for the D124N mutant and to >100-fold in the K354M mutant. This result is better visualized in Fig.3, in which the two observed rate constants of heme a3 reduction for both mutants are reported at different NO concentrations (from 0 to 500 μm). The data show that internal electron transfer in the D124N mutant is faster than in the K354M mutant. Moreover, in both mutants, the two rate constants (relative to the fast and the slow kinetic phases) depend on the NO concentration. Although the dependence is much less pronounced in the D124N mutant, all rate constants become essentially independent of [NO], reaching plateau values atk 1 = 13 ± 6 s−1 andk 2 = 0.7 ± 0.3 s−1 in the K354M mutant and k 1 = 110 ± 16 s−1 and k 2 = 3.5 ± 1.5 s−1 in the D124N mutant. All asymptotic values are independent of reductant concentration, as assessed by experiments at 500 μm NO and variable ruthenium hexamine concentration; at ruthenium hexamine concentrations above 1 mm (after mixing), the observed rate constants were independent of reductant concentration.Figure 2Reduction of the D124N aa3 and the effect of NO. The oxidized D124N mutant of the P. denitrificans cytochrome oxidase is anaerobically mixed with reductants in the absence (A) and in the presence of NO (B). Experimental conditions are as described in the legend for Fig. 1. A and B, similar to the K354Maa 3, heme a reduction is complete within a few milliseconds, independently of NO. Afterward, complete reduction of the enzyme is achieved within a few seconds in the absence of NO (A) and on an even shorter time scale in the presence of NO (B). C, time courses of heme a3reduction as obtained by the pseudoinverse analysis. In the absence of NO, heme a3 is reduced with rate constants ofk 1 = 15 s−1 (60% total amplitude) and k 2 = 2.7 s−1 (40% total amplitude). At 500 μm NO, heme a3 reduction is faster and proceeds with rate constants of k 1= 78 s−1 (60% total amplitude) andk 2 = 5.1 s−1 (40% total amplitude).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of NO concentration. Rate constants of heme a3 reduction, relative to the fast (top) and the slow (bottom) phases, were measured at varying NO concentrations. Experimental conditions were as described in the legend for Fig. 1. Under all conditions, internal electron transfer in the D124N mutant is faster than in the K354M mutant. Both rate constants depend on the NO concentration, although the dependence is much less pronounced in the D124N mutant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It is worth noticing that the faster heme a3 reduction observed with both mutants in the presence of NO is not due to direct reduction of the oxidized binuclear center O by NO. As demonstrated with beef heart CcOX and confirmed with the P. denitrificans wild-type CcOX, the reaction of NO with Ooccurs rapidly with the chloride-free enzyme, yielding nitrite-bound heme a3 and reduced heme a (25Cooper C.E. Torres J. Sharpe M.A. Wilson M.T. FEBS Lett. 1997; 414: 281-284Crossref PubMed Scopus (106) Google Scholar), but it is prevented with the chloride-bound oxidase (26Giuffrè 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). Spectrophotometrically, we did not detect a reaction after mixing both oxidized mutants with NO (1 mm after mixing, not shown). Further in this respect, the reactivity of NO with the oxidized K354M oxidase was probed by amperometry by using a NO-selective Clark-type electrode (24Stubauer G. Giuffrè A. Brunori M. Sarti P. Biochem. Biophys. Res. Commun. 1998; 245: 459-465Crossref PubMed Scopus (67) Google Scholar). If the oxidized enzyme is anaerobically added to a degassed NO-containing solution, a reaction would be detected as a decrease in the NO concentration. Upon the addition of oxidized K354M CcOX (0.4 μm) to a solution containing NO (1 μm), only a small decrease in the NO concentration (∼0.2 mol of NO/mol of oxidase) was detected; in contrast, a stoichiometric (1:1) NO binding was observed after the addition of the fully reduced enzyme, as shown for the mammalian CcOX (24Stubauer G. Giuffrè A. Brunori M. Sarti P. Biochem. Biophys. Res. Commun. 1998; 245: 459-465Crossref PubMed Scopus (67) Google Scholar). We therefore conclude that both theP. denitrificans mutants tested in this study are in the chloride-bound state, due to the presence of chloride in the buffers used during both the purification and the experiments.DISCUSSIONNO is a very efficient, yet reversible, inhibitor of cytochromec oxidase activity (27Brown G.C. Cooper C.E. FEBS Lett. 1994; 356: 295-298Crossref PubMed Scopus (934) Google Scholar, 28Cleeter M.W.J. Cooper J.M. Darley-Usmar V.M. Moncada S. Schapira A.H.V. FEBS Lett. 1994; 345: 50-54Crossref PubMed Scopus (1144) Google Scholar), leading to the proposal that it may act as a physiological modulator of cell respiration (29Brown G.C. FEBS Lett. 1995; 369: 136-139Crossref PubMed Scopus (502) Google Scholar). Since both NO and O2 react with the fully reduced heme a3-CuB center R with high affinity and similar rates (19Gibson Q.H. Greenwood C. Biochem. J. 1963; 86: 541-555Crossref PubMed Scopus (268) Google Scholar), the small inhibition constantK i determined with mitochondrial CcOX in turnover (K i = 270 nm NO at [O2] = 140 μm, (27Brown G.C. Cooper C.E. FEBS Lett. 1994; 356: 295-298Crossref PubMed Scopus (934) Google Scholar)) was somewhat puzzling. To account for this observation, it was proposed that NO can react with a single-electron reduced active site E (20–21), which is known to be unreactive toward O2. Such a hypothesis is consistent with computer simulations (20Torres J. Darley-Usmar V.M. Wilson M.T. Biochem. J. 1995; 312: 169-173Crossref PubMed Scopus (185) Google Scholar, 21Giuffrè A. Sarti P. D'Itri E. Buse G. Soulimane T. Brunori M. J. Biol. Chem. 1996; 271: 33404-33408Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) but has never been demonstrated experimentally. The kinetics of reduction of the K354M mutant ofP. denitrificans in the presence of NO, reported above, provides evidence that this hypothesis is correct.The K354M mutation is associated with the loss of oxidase activity (5Pfitzner U. Odenwald A. Ostermann T. Weingard L. Ludwig B. Richter O.M. J. Bioenerg. Biomembr. 1998; 30: 89-97Crossref PubMed Scopus (115) Google Scholar, 9Hosler J.P. Shapleigh J.P. Mitchell D.M. Kim Y. Pressler M.A. Georgiou C. Babcock G.T. Alben J.O. Ferguson-Miller S. Gennis R.B. Biochemistry. 1996; 35: 10776-10783Crossref PubMed Scopus (89) Google Scholar, 11Jünemann S. Meunier B. Gennis R.B. Rich P.R. Biochemistry. 1997; 36: 14456-14464Crossref PubMed Scopus (94) Google Scholar), although this mutant in the fully reduced state (R) is very quickly (∼5 ms) oxidized by O2, as reported for the R. sphaeroides CcOX (15, but see also Ref.13Brändén M. Sigurdson H. Namslauer A. Gennis R.B. Ädelroth P. Brzezinski P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5013-5018Crossref PubMed Scopus (136) Google Scholar). The same mutation has a dramatic effect on the reductive part of the catalytic cycle, and the extremely low rate of reduction of heme a3 was correlated to the marginal turnover rate (9Hosler J.P. Shapleigh J.P. Mitchell D.M. Kim Y. Pressler M.A. Georgiou C. Babcock G.T. Alben J.O. Ferguson-Miller S. Gennis R.B. Biochemistry. 1996; 35: 10776-10783Crossref PubMed Scopus (89) Google Scholar, 11Jünemann S. Meunier B. Gennis R.B. Rich P.R. Biochemistry. 1997; 36: 14456-14464Crossref PubMed Scopus (94) Google Scholar,14Zaslavsky D. Gennis R.B. Biochemistry. 1998; 37: 3062-3067Crossref PubMed Scopus (45) Google Scholar). It was therefore assumed that in this mutant, the turnover with O2 is rate-limited by the extremely slow formation ofR, which is an obligatory intermediate in the catalytic cycle. This is consistent with the widely accepted idea that O2 can react exclusively with the two-electron reduced heme a3-CuB site, like CO.The novel result reported in this study on the K354M mutant of P. denitrificans CcOX is that, in the presence of NO, the reduction of heme a3 occurs at a rate much faster (>100-fold) than in its absence and much faster than the extremely low turnover rate of this mutant with O2 (Fig. 1). This effect depends on NO concentration and is maximal at [NO] >100 μm (Fig. 3), at which the reduction of heme a3 proceeds at rates (k 1 = 13 ± 6 s−1;k 2 = 0.7 ± 0.3 s−1) both remarkably larger than the turnover rate (∼0.02 mol of O2/mol of CcOX × s at [O2] > 250 μm). This finding is diagnostic of a different reactivity of O2 and NO with CcOX. It is indeed difficult to account for this result, assuming that NO, similarly to O2, can react exclusively with R, which combines with very high affinity and second order rate constants with both ligands. If this were the case, the formation of the reduced NO-bound heme a3 would be much slower, being rate-limited by the formation of R, which in turn accounts for the extremely low oxidase activity. Therefore this result implies that NO can react not only with the two-electron reduced heme a3-CuBsite R (19) but also with the single-electron reduced siteE, whose occurrence in this mutant was already documented (11Jünemann S. Meunier B. Gennis R.B. Rich P.R. Biochemistry. 1997; 36: 14456-14464Crossref PubMed Scopus (94) Google Scholar). We do not have a valid explanation for the observed heterogeneity in the reduction of heme a3, but we notice that a similar biphasic behavior was reported also for the beef heart enzyme (7Verkhovsky M.I. Morgan J.E. Wikström M. Biochemistry. 1995; 34: 7483-7491Crossref PubMed Scopus (117) Google Schol" @default.
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