Structures of reaction intermediates of bovine cytochrome c oxidase probed by time-resolved vibrational spectroscopy

Structures of reaction intermediates of bovine cytochrome c oxidase (CcO) in the reactions of its fully reduced form with O2 and fully oxidized form with H2O2 were investigated with time-resolved resonance Raman (RR) and infrared spectroscopy. Six oxygen-associated RR bands were observed for the reaction of CcO with O2. The isotope shifts for an asymmetrically labeled dioxygen, (16)O(18)O, has established that the primary intermediate of cytochrome a3 is an end-on type dioxygen adduct and the subsequent intermediate (P) is an oxoiron species with Fe=O stretch (nu(Fe=O)) at 804/764 cm(-1) for (16)O2/(18)O2 derivatives, although it had been long postulated to be a peroxy species. The P intermediate is converted to the F intermediate with nu(Fe=O) at 785/751 cm(-1) and then to a ferric hydroxy species with nu(Fe-OH) at 450/425 cm(-1) (443/417 cm(-1) in D2O). The rate of reaction from P to F intermediates is significantly slower in D2O than in H2O. The reaction of oxidized CcO with H2O2 yields the same oxygen isotope-sensitive bands as those of P and F, indicating the identity of intermediates. Time-resolved infrared spectroscopy revealed that deprotonation of carboxylic acid side chain takes place upon deligation of a ligand from heme a3. UV RR spectrum gave a prominent band due to cis C=C stretch of phospholipids tightly bound to purified CcO.     

ATR-FTIR spectroscopy and isotope labeling of the PM intermediate of Paracoccus denitrificans cytochrome c oxidase

The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes. In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site. In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments. In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine. In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected. Both isotope substitutions had large effects on P(M) minus oxidized difference spectra. A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling. Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra. These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B). Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate. Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M).     

Structural and chemical changes of the P(M) intermediate of paracoccus denitrificans cytochrome c oxidase revealed by IR spectroscopy with labeled tyrosines and histidine

Structural and chemical changes in the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase have been investigated by attenuated total reflection-Fourier transform infrared spectroscopy. Prior studies of P(M) minus oxidized (O) IR difference spectra of unlabeled, universally (15)N-labeled and ring-d(4)-tyrosine-labeled proteins (Iwaki, M., Puustinen, A., Wikstr?m, M., and Rich, P. R. (2004) Biochemistry 43, 14370-14378). provided a basis for band assignments to changes in metal centers and the covalently linked His-Tyr ligand of Cu(B) and highlighted a structural alteration of the protonated Glu278 in the P(M) intermediate. This work has been extended to equivalent measurements on enzymes with (13)C(9)(15)N-labeled and ring-(13)C(6)-labeled tyrosine and with (13)C(6)(15)N(3)-labeled histidine. Histidine labeling allows the assignment of troughs at 1104 and 973 cm(-1) in reduced minus O spectra to histidine changes, whereas tyrosine labeling moves otherwise obscured tyrosine bandshifts to 1454-1437 and 1287-1284 cm(-1). P(M) minus O spectra reveal bands at 1506, 1311, and 1094 cm(-1) in the oxidized state that are replaced by a band at 1519 cm(-1) in P(M). These bands shift with both tyrosine- and histidine-labeling, providing evidence for their assignment to the covalent His-Tyr and for its chemical change in P(M). Comparisons of isotope effects on the amide I regions in P(M) minus O spectra demonstrate that amide carbonyl bonds of tyrosine and histidine are major contributors. This suggests a structural alteration in P(M) that is centered on the His276-Pro277-Glu278-Val279-Tyr280 pentapeptide formed by the His-Tyr covalent linkage. This structural change is proposed to mediate the perturbation of the IR band of the protonated Glu278 headgroup.     

Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2). The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode. Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo.     

Resonance Raman characterization of the P intermediate in the reaction of bovine cytochrome c oxidase

Reduced cytochrome c oxidase binds molecular oxygen, yielding an oxygenated intermediate first (Oxy) and then converts it to water via the reaction intermediates of P, F, and O in the order of appearance. We have determined the iron-oxygen stretching frequencies for all the intermediates by using time-resolved resonance Raman spectroscopy. The bound dioxygen in Oxy does not form a bridged structure with Cu(B) and the rate of the reaction from Oxy to P (P(R)) is slower at higher pH in the pH range between 6.8 and 8.0. It was established that the P intermediate has an oxo-heme and definitely not the Fe(a(3))-O-O-Cu(B) peroxy bridged structure. The Fe(a(3))=O stretching (nu(Fe=O)) frequency of the P(R) intermediate, 804/764 cm(-1) for (16)O/(18)O, is distinctly higher than that of F intermediate, 785/750 cm(-1). The rate of reaction from P to F in D(2)O solution is evidently slower than that in H(2)O solution, implicating the coupling of the electron transfer with vector proton transfer in this process. The P intermediate (607-nm form) generated in the reaction of oxidized enzyme with H(2)O(2) gave the nu(Fe=O) band at 803/769 cm(-1) for H(2)(16)O(2)/H(2)(18)O(2) and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O(2) provided the P intermediate (P(M)) giving rise to an absorption peak at 607 nm and the nu(Fe=O) bands at 804/768 cm(-1). Thus, three kinds of P intermediates are considered to have the same oxo-heme a(3) structure. The nu(4) and nu(2) modes of heme a(3) of the P intermediate were identified at 1377 and 1591 cm(-1), respectively. The Raman excitation profiles of the nu(Fe=O) bands were different between P and F. These observations may mean the formation of a pi cation radical of porphyrin macrocycle in P.     

Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans. Evidence from FTIR difference spectroscopy and site-directed mutagenesis

By specific (13)C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates [Behr, J., Hellwig, P., M?ntele, W., and Michel, H. (1998) Biochemistry 37, 7400-7406]. To attribute these signals to the individual propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I. The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromic oxidase. The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor.     

Effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c on reactions with oxidase, reductase and peroxidase

The effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c (Kuo, L.M. and Davies, H.C. (1983) Mol. Immunol. 20, 827-838) in the reactions of the cytochromes c with cytochrome c oxidase, reductase and peroxidase were studied. Spectrophotometric assays were employed, under conditions where binding of cytochrome c to the enzymes appears to be rate-limiting. Less than stoichiometric amounts of antibodies to P. denitrificans cytochrome c added to the cytochrome rendered some of it nonoxidizable or nonreducible by the P. denitrificans membrane-bound electron transport system and decreased the rate constant with the remaining cytochrome c. The antibodies appear to affect both electron transport reactions (blocking effects) with the oxidase and reductase and binding effects (effects on rate constants) and to distinguish between the two. Different ratios of antibody site to cytochrome c gave different extents of blocking of the reductase as compared with the oxidase reaction. Differences were also apparent in the effect of these antibodies on the reaction of yeast peroxidase and the oxidase with the P. denitrificans cytochrome c. Antibodies to bovine and P. denitrificans cytochromes c had considerably less effect on the reactions of the bovine cytochrome with bovine oxidase and reductase. One antibody was inhibitory to the oxidase reaction with bovine cytochrome c, but not to that with the reductase. Also, an antibody which inhibited the oxidase reaction had no effect on the reaction with yeast peroxidase. The data give evidence that the interaction areas on cytochrome c for oxidase and reductase and peroxidase are not identical, although they may be nearby.     

The reactions of hydrogen peroxide with bovine cytochrome c oxidase

Oxidised cytochrome c oxidase is known to react with two molecules of hydrogen peroxide to form consecutively 607 nm 'Peroxy' and 580-nm 'Ferryl' species. These are widely used as model compounds for the equivalent P and F intermediates of the catalytic cycle. However, kinetic analysis of the reaction with H(2)O(2) in the pH range 6.0-9.0 reveals a more complex situation. In particular, as the pH is lowered, a 580-nm compound can be formed by reaction with a single H(2)O(2). This species, termed F(&z.rad;), is spectrally similar, but not identical, to F. The reactions are equivalent to those previously reported for the bo type quinol oxidase from Escherichia coli (T. Brittain, R.H. Little, C. Greenwood, N.J. Watmough, FEBS Lett. 399 (1996) 21-25) where it was proposed that F(&z.rad;) is produced directly from P. However, in the bovine oxidase F(&z.rad;) does not appear in samples of the 607-nm form, P(M), produced by CO/O(2) treatment, even at low pH, although this form is shown to be identical to the H(2)O(2)-derived P state, P(H), on the basis of spectral characteristics and kinetics of reaction with H(2)O(2). Furthermore, lowering the pH of a sample of P(M) or P(H) generated at high pH results in F(&z.rad;) formation only on a minutes time scale. It is concluded that P and F(&z.rad;) are not in a rapid, pH-dependent equilibrium, but instead are formed by distinct pathways and cannot interconvert in a simple manner, and that the crucial difference between them lies in their patterns of protonation.     

Allosteric interactions and proton conducting pathways in proton pumping aa(3) oxidases: heme a as a key coupling element

In this paper allosteric interactions in protonmotive heme aa(3) terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H(+)/e(-) coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa(3) oxidase, which decreases by more than 200mV the E(m) of heme a, inhibits proton pumping. Mutational amino acid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa(3) oxidases, as well as Zn(2+) binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O(2) to 2 H(2)O.     

ATR-FTIR difference spectroscopy of the P(M) intermediate of bovine cytochrome c oxidase

Perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate changes induced in protein and cofactors of bovine cytochrome c oxidase when it was converted from the oxidised state to the catalytic P(M) intermediate. The transition was induced in a film of detergent-depleted 'fast' oxidase with a buffer containing CO and O(2). The extent of formation of the P(M) state was quantitated simultaneously by monitoring formation of its characteristic 607-nm band with a scanned visible beam reflected off the top surface of the prism. The P(M) minus O FTIR difference spectrum is distinctly different from the redox spectra reported to date and includes features that can be assigned to changes of haem a(3) and surrounding protein. Tentative assignments are made based on vibrational data of related proteins and model compounds.     

Single-electron photoreduction of the P(M) intermediate of cytochrome c oxidase

The P(M)-->F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P(M) state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is approximately 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F-->O transition, but is significantly slower than formation of the F state from the P(R) intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70-90 micros). The approximately 0.3 ms P(M)-->F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P(M)-->F transition is generated much later, with a time constant of 1.3 ms. In addition, the P(M)-->F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with tau values of 0.18 and 0.85 ms.     

ATR-FTIR redox difference spectroscopy of Yarrowia lipolytica and bovine complex I

ATR-FTIR spectroscopy in combination with electrochemistry has been applied to the redox centers of Yarrowia lipolytica complex I. The redox spectra show broad similarities with previously published data on Escherichia coli complex I and with new data here on bovine complex I. The spectra are dominated by amide I/II protein backbone changes. Comparisons with redox IR spectra of small model ferredoxins demonstrate that these amide I/II changes arise primarily from characteristic structural changes local to the iron-sulfur centers, rather than from global structural alterations as has been suggested previously. Bands arising from the substrate ubiquinone were evident, as was a characteristic 1405 cm(-)(1) band of the reduced form of the FMN cofactor. Other signals are likely to arise from perturbations or protonation changes of a carboxylic amino acid, histidine, and possibly several other specific amino acids. Redox difference spectra of center N2, together with substrate ubiquinone, were isolated from those of the other iron-sulfur centers by selective redox potentiometry. Its redox-linked amide I/II changes were typical of those in other 4Fe-4S iron sulfur proteins. Contrary to published data on bacterial complex I, no center N2 redox-linked protonation changes of carboxylic amino acids or tyrosine were evident, and other residues that could provide its redox-linked protonation site are discussed. Features of the substrate ubiquinone associated with the center N2 spectrum were particularly clear, with firm assignments possible for bands from both oxidized and reduced forms. This is the first report of IR properties of ubiquinone in complex I, and the data could be used to estimate a stoichiometry of 0.2-0.4 per complex I.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P(M), F and F(z.rad;)) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F(z.rad;) but when F(z.rad;) is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P(M) is produced at high pH with CO/O(2), no EPR-detectable radicals are formed. Conversion of the CO/O(2)-generated P(M) into F(z.rad;) when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F(z.rad;) determined optically. It is concluded that low pH causes a change of protonation pattern in P(M) which induces partial electron redistribution and tryptophan cation radical formation in F(z.rad;). These protonation changes may mimic a key step of the proton translocation process.     

Monitoring the pH dependence of IR carboxylic acid signals upon Q(B)- formation in the Glu-L212 --> Asp/Asp-L213 --> Glu swap mutant reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (QB) site. Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations in the 1770-1700 cm-1 spectral range that are sensitive to 1H/2H isotopic exchange. With respect to the native RC, a novel protonation pattern for carboxylic acids upon QB photoreduction has been identified in the Glu-L212 --> Asp/Asp-L213 --> Glu mutant RC using light-induced FTIR difference spectroscopy (Nabedryk, E., Breton, J., Okamura, M. Y., and Paddock, M. L. (2004) Biochemistry 43, 7236-7243). These carboxylic acids are structurally close and have been implicated in proton transfer to reduced QB. In this work, we extend previous studies by measuring the pH dependence of the QB-/QB FTIR difference spectra of the mutant in 1H2O and 2H2O. Large pH dependent changes were observed in the 1770-1700 cm-1 spectral range between pH 8 and pH 4. The IR fingerprints of the protonating carboxylic acids upon QB- formation were obtained from the calculated double-difference spectra 1H2O minus 2H2O. These IR fingerprints are specific for each pH, indicative of the contribution of different titrating groups. In particular, the 1752 cm-1 signal indicates that Glu-L213 protonates upon QB- formation at pH >or= 5, whereas the 1746 cm-1 signal indicates protonation of Asp-L212 even at pH 4. An unidentified carboxylic acid absorbing at approximately 1765 cm-1 could be the proton donor between pH 8 and 5. The observation that in the swap mutant there are several uniquely behaving carboxylic acids shows that electrostatic interactions occurring between them are sufficiently modified from the native RC to reveal their IR signatures.     

FTIR studies of the CO and cyanide adducts of fully reduced bovine cytochrome c oxidase

Photolysis spectra of the CO and cyanide adducts of reduced bovine cytochrome c oxidase have been studied by FTIR difference spectroscopy. Bound CO is predominantly in a single 1963 cm(-1) form whereas cyanide is bound in at least two forms (2058/2045 cm(-1)). These forms are pH-independent between pH 6.5 and 8.5, indicating that there is no titratable protonatable group that influences significantly their binding in this pH range. Photolysis spectra of the cyanide adduct have a positive band around 2090 cm(-1) in H(2)O due at least in part to free HCN and at 1880 cm(-1) in D(2)O due to free DCN. The frequency of the positive band around 2090 cm(-1), and its persistence in D(2)O media, raises the possibility that a transient cyanide-Cu(B) adduct also contributes to this signal, equivalent to the CO-Cu(B) species that is formed when CO is photolyzed. Photolysis produces changes throughout the 1000-1800 cm(-1) region. Reduced minus (reduced + CO) photolysis spectra in H(2)O exhibit a pH-independent and symmetrical peak/trough at 1749/1741 cm(-1). A related feature in homologous oxidases has been suggested to arise from a conserved glutamic acid. However, only around one-third of the feature is shifted to lower frequencies by incubation in D(2)O media, and an additional fraction is shifted if catalytic turnover occurs in D(2)O. Reduced minus (reduced + cyanide) photolysis spectra exhibit multiple features in H(2)O in this region with peaks at 1752, 1725, and 1708 cm(-1) and troughs at 1740, 1715, and 1698 cm(-1). Again, only a part of these features shift in D(2)O, even with catalytic turnover. A variety of additional H/D-sensitive features in the 1700-1000 cm(-1) region of the spectra can be discerned, one of which in cyanide photolysis spectra is tentatively assigned to a conserved tyrosine, Y244. Data are discussed in relation to the structure of the binuclear center and protonatable groups in its vicinity.     

Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: comparison of the fully reduced and the mixed valence form.

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.     

Structural characterization of the [Pco/o(2)] compound of cytochrome c oxidase

The structural properties of a key transient oxygen intermediate of cytochrome c oxidase, P(R), remain an enigma, although inferences have been drawn from its equilibrium analogues, [Pco/o(2)] , P(H) and P(M). With resonance Raman spectroscopy, an oxygen isotope-sensitive band at 806 cm(-1) was observed in [Pco/o(2)] produced by adding CO and O(2) to the resting enzyme. The vibrational band shifted to 771 cm(-1) upon isotopic substitution of (16)O(2) with (18)O(2). The same modes at 806 and 771 cm(-1) were present simultaneously when the mixed isotope, (18)O(16)O, was employed, indicating that in [Pco/o(2)] the O-O bond is cleaved, resulting in a Fe(4+)O(2-) structure. This result unifies the nature of the three equilibrium analogues of the P(R) intermediate.     

Cytochrome c oxidase as a calcium binding protein. Studies on the role of a conserved aspartate in helices XI-XII cytoplasmic loop in cation binding

The aa(3)-type cytochrome c oxidases from mitochondria and bacteria contain a cation-binding site located in subunit I near heme a. In the oxidases from Paracoccus denitrificans or Rhodobacter sphaeroides, the site is occupied by tightly bound calcium, whereas the mitochondrial oxidase binds reversibly calcium or sodium that compete with each other. The functional role of the site has not yet been established. D477A mutation in subunit I of P. denitrificans oxidase converts the cation-binding site to a mitochondrial-type form that binds reversibly calcium and sodium ions [Pfitzner, U., Kirichenko, A., et al. (1999) FEBS Lett. 456, 365-369]. We have studied reversible cation binding with P. denitrificans D477A oxidase and compared it with that in bovine enzyme. In bovine oxidase, one Ca(2+) competes with two Na(+) for the binding, indicating the presence of two Na(+)-binding sites in the enzyme, Na(+)((1)) and Na(+)((2)). In contrast, the D477A mutant of COX from P. denitrificans reveals competition of Ca(2+) (K(d) = 1 microM) with only one sodium ion (K(d) = 4 mM). The second binding site for Na(+) in bovine oxidase is proposed to involve D442, homologous to D477 in P. denitrificans oxidase. A putative place for Na(+)((2)) in subunit I of bovine oxidase has been found with the aid of structure modeling located 7.4 A from the bound Na(+)((1)) . Na(+)((2)) interacts with a cluster of residues forming an exit part of the so-called H-proton channel, including D51 and S441.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Redox-induced transitions in bovine cytochrome bc1 complex studied by perfusion-induced ATR-FTIR spectroscopy

Redox transitions in a film of detergent-purified bovine cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The technique provides a flexible method for generating redox-induced IR changes of components of bovine cytochrome bc(1) complex at a high signal:noise ratio. These IR redox difference spectra arise from perturbations of prosthetic groups and surrounding protein. Visible difference spectra were recorded synchronously using a light beam reflected from the exposed prism surface and provided a quantitative means of determining the redox transitions that were occurring. IR and visible redox difference spectra of iron-sulfur protein/cytochrome c(1), heme b(H), and heme b(L) were separated by selective reduction and/or oxidation that extends published data on the homologous bacterial enzyme. Several bands could be tentatively assigned to redox-sensitive modes of hemes and ubiquinone and changes in the surrounding protein by comparison with available data for bacterial bc(1) complex, other related heme proteins, and model compounds. Some tentative assignments of further signals to specific amino acids are made on the basis of known crystal structures.