Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects.

The reactions of nitric oxide (NO) with the turnover intermediates of cytochrome c oxidase were investigated by combining amperometric and spectroscopic techniques. We show that the complex of nitrite with the oxidized enzyme (O) is obtained by reaction of both the "peroxy" (P) and "ferryl" (F) intermediates with stoichiometric NO, following a common reaction pathway consistent with P being an oxo-ferryl adduct. Similarly to chloride-free O, NO reacted with P and F more slowly [k approximately (2-8) x 10(4) M(-1) s(-1)] than with the reduced enzyme (k approximately 1 x 10(8) M(-1) s(-1)). Recovery of activity of the nitrite-inhibited oxidase, either during turnover or after a reduction-oxygenation cycle, was much more rapid than nitrite dissociation from the fully oxidized enzyme (t(1/2) approximately 80 min). The anaerobic reduction of nitrite-inhibited oxidase produced the fully reduced but uncomplexed enzyme, suggesting that reversal of inhibition occurs in turnover via nitrite dissociation from the cytochrome a(3)-Cu(B) site: this finding supports the hypothesis that oxidase may have a physiological role in the degradation of NO into nitrite. Kinetic simulations suggest that the probability for NO to be transformed into nitrite is greater at low electron flux through oxidase, while at high flux the fully reduced (photosensitive) NO-bound oxidase is formed; this is fully consistent with our recent finding that light releases the inhibition of oxidase by NO only at higher reductant pressure [Sarti, P., et al. (2000) Biochem. Biophys. Res. Commun. 274, 183].

The ratio between the fast and slow forms of bovine cytochrome c oxidase is changed by cholate or nucleotides bound to the cholate-binding site close to the cytochrome a3/CuB binuclear centre.

We determined the fraction of 'slow' and 'fast' conformations of bovine cytochrome c oxidase, following the kinetics of cyanide binding to the oxidized enzyme. We investigated whether treatment of heart mitochondrial particles with different commercially available types of cholate (standard and ultrapure) can affect the fraction of cytochrome c oxidase in the two states. Compared to standard cholate, the use of ultra-pure cholate for solubilization of heart mitochondrial particles significantly increased the fraction of the fast enzyme. Complete homogeneity (approximately 100% fast) was observed when cytochrome c oxidase was solubilized with ultra-pure cholate from heart mitochondrial particles pre-equilibrated with AMP; equilibration with ADP yielded a much smaller fraction of fast enzyme (approximately 35%). These observations are discussed on the basis of the structural relationships between the known cholate-binding site and the binuclear cytochrome a3-CuB site: variation in the occupancy of this binding site with cholate or nucleotides may modify reactivity of the oxidized binuclear centre towards cyanide.

Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature.

The kinetic properties of the ba3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70 degrees C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O2 reaction, the decay of the F intermediate is significantly slower (k' = 100 s-1 at 5 degrees C) than in the mitochondrial enzyme, with an activation energy E of 10.1 +/- 0.9 kcal mol-1. The cyanide-inhibited ba3 oxidizes cyt c522 quickly (k approximately 5 x 10(6) M-1 s-1 at 25 degrees C) and selectively, with an activation energy E of 10.9 +/- 0.9 kcal mol-1, but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c552 oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c522-ba3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba3 for CO at 20 degrees C (Keq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa3 (Keq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a32+ of ba3 (k' = 0.8 s-1 vs k' = 0.02 s-1 for beef heart aa3 at 20 degrees C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O2 tensions is discussed.

Kinetic control of internal electron transfer in cytochrome c oxidase.

Two alternative hypotheses have been proposed to account for the relatively slow (ms) internal eT observed in the oxidized cyt c oxidase. The thermodynamic control hypothesis states that eT between cyt a and a3 is very fast (microsecond), but the apparent reduction of cyt a3 is slow because thermodynamics favors reduced cyt a. Whereas the kinetic control hypothesis states that inter-heme eT is intrinsically slow (ms), for the oxidized binuclear center. Monitoring by stopped flow the anaerobic reduction of the oxidized enzyme by ruthenium hexamine in the absence and presence of CO or NO, used as "trapping" ligands for cyt a3(2+), we found that the rate of formation of the cyt a3(2+)-NO adduct (k' approximately 20-25 s-1) is independent of the concentration of ruthenium hexamine and NO. We conclude that in the oxidized enzyme the two hemes are not in very rapid redox equilibrium and internal eT is kinetically controlled.

Internal electron transfer in Cu-heme oxidases. Thermodynamic or kinetic control?

We present novel experimental evidence that, starting with the oxidized enzyme, the internal electron transfer in cytochrome c oxidase is kinetically controlled. The anaerobic reduction of the oxidized enzyme by ruthenium hexamine has been followed in the absence and presence of CO or NO, used as trapping ligands for reduced cytochrome a3. In the presence of NO, the rate of formation of the cytochrome a32+-NO adduct is independent of the concentration of ruthenium hexamine and of NO, indicating that in the oxidized enzyme cytochrome a and a3 are not in very rapid redox equilibrium; on the other hand, CO proved to be a poor "trapping" ligand. We conclude that the intrinsic rate constant for a --> a3 electron transfer in the oxidized enzyme is 25 s-1. These data are discussed with reference to a model (Verkhovsky, M. I., Morgan, J. E., and Wikström, M. (1995) Biochemistry 34, 7483-7491) in which H+ diffusion and/or binding at the binuclear site is the rate-limiting step in the reduction of cytochrome a3 in the oxidized enzyme.

Functional properties of the quinol oxidase from Acidianus ambivalens and the possible catalytic role of its electron donor--studies on the membrane-integrated and purified enzyme.

The aa3 quinol oxidase has been purified from the thermoacidophilic archaea Acidianus ambivalens as a three-redox-centers enzyme. The functional properties of this oxidase both as purified and in its most integral form (i.e. in native membranes and in intact cells) were investigated by stopped-flow spectrophotometry. The results suggest that the enzyme interacts in vivo with a redox-active molecule, which favours the electron entry via heme a and provides the fourth electron demanded for catalysis. We observe that the purified enzyme has two hemes with apparent redox potentials 215 +/- 20 mV and 415 +/- 20 mV at pH 5.4, showing redox-Bohr effect, and a heme a3-CuB center with an affinity for carbon monoxide (Ka = 5.7 x 10(4) M(-1) at 35 degrees C) much lower than that reported for the mammalian enzyme (Ka = 4 x 10(6) M(-1) at 20 degrees C). The reduction by dithionite is fast and monophasic when the quinol oxidase is in the native membranes, whereas it is slow and biphasic in the purified enzyme (with heme a3 being reduced faster than heme a). The oxygen reaction of the reduced purified enzyme is fast (few milliseconds), but yields an intermediate (likely ferryl) clearly different from the fully oxidized enzyme. In contrast, the same reaction performed in intact cells leads to the fully oxidized enzyme. We postulate that caldariella quinol, the physiological electron donor, is in vivo tightly bound to the enzyme, providing the fourth redox active center lacking in the purified enzyme.

On the mechanism of inhibition of cytochrome c oxidase by nitric oxide.

The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric oxide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis. Following the time course of cyt c oxidation at different O2/NO ratios, we observed that the onset of inhibition: (i) is fast and at a high NO concentration is complete during the first turnover; (ii) is sensitive to the O2/NO ratio; and (iii) is independent of incubation time of the oxidized enzyme with NO. Analysis of the reaction kinetics and computer simulations support the conclusion that inhibition occurs via binding of NO to a turnover intermediate with a partially reduced cyt a3-CuB binuclear center. The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a3. Reversal of inhibition in the presence of O2 does not involve a direct reaction of O2 with NO while bound at the binuclear center, since recovery of activity occurs at the rate of NO dissociation (k = 0.13 s-1), as determined in the absence of O2 using hemoglobin as a NO scavenger. We propose that removal of NO from the medium is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a3-CuB center.

The caa3 terminal oxidase of Bacillus stearothermophilus. Transient spectroscopy of electron transfer and ligand binding.

The thermophilic bacterium Bacillus stearothermophilus possesses a caa3-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R. I. R., and Konings, W. N. (1989) Eur. J. Biochem. 178, 763-770). We have carried out extensive kinetic experiments on the purified enzyme by stopped-flow time-resolved optical spectroscopy combined with singular value decomposition analysis. The results indicate a striking similarity of behavior between this enzyme and the electrostatic complex between mammalian cytochrome c and cytochrome c oxidase. CO binding to fully reduced caa3 occurs with a second order rate constant (k = 7.8 x 10(4)M-1 s-1) and an activation energy (E* = 6.1 kcal mol-1) similar to those reported for beef heart cytochrome c oxidase. Dithionite reduces cytochrome a with bimolecular kinetics, while cytochrome a3 (and CuB) is reduced via intramolecular electron transfer. When the fully reduced enzyme is mixed with O2, cytochrome a3, and cytochrome c are rapidly oxidized, whereas cytochrome a remains largely reduced in the first few milliseconds. When cyanide-bound caa3 is mixed with ascorbate plus TMPD, cytochrome c and cytochrome a are synchronously reduced; the value of the second order rate constant (k = 3 x 10(5) M-1 s-1 at 30 degrees C) suggests that cytochrome c is the electron entry site. Steady-state experiments indicate that cytochrome a has a redox potential higher than cytochrome c. The data from the reaction with O2 reveal a remarkable similarity in the kinetic, equilibrium, and optical properties of caa3 and the electrostatic complex cytochrome c/cytochrome c oxidase.

Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking.

A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa3) has been prepared at low ionic strength. Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate [Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046-7053]. Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa3 are higher than those found in the native aa3 enzyme. However, (3) K(m) and Vmax values obtained from the non-linear Eadie-Hofstee plots are very similar in both caa3 and aa3. The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.

Sulfolobus acidocaldarius terminal oxidase. A kinetic investigation and its structural interpretation.

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius possesses a very unusual terminal oxidase. We report original kinetic experiments on membranes of this microorganism carried out by stopped flow, using time-resolved optical spectroscopy combined with singular value decomposition analysis. The reduced-oxidized kinetic difference spectrum of the Sulfolobus membranes is characterized by three significant peaks in the visible region at 605, 586, and 560 nm. The 605-nm peak and part of the 586-nm peak (cytochrome aa3-type quinol oxidase) are reduced synchronously by both ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and dithionite, and they are very rapidly oxidized by molecular oxygen. A second pool of cytochromes seems to contribute to the 586-nm peak which is not reduced by ascorbate plus TMPD and reacts very slowly with dithionite. The b-type cytochromes (560 nm peak) are reduced by both reductants and are essentially "non-autoxidizable" at room temperature. Only one CO binding site with spectral features, kinetic properties, and ligand affinity not very dissimilar from those of mammalian cytochrome oxidase can be detected in the ascorbate-reduced membranes. On the contrary, a second CO binding site having unusual properties for aa3 terminal oxidases can be detected in the dithionite-reduced membranes.

Spectral analysis of cytochromes in rat heart myocytes: transient and steady-state photodiode array spectrophotometry measurements.

Myocytes prepared from rat heart have been studied by optical spectroscopy using a photodiode array spectrophotometer adapted to a stopped flow apparatus (PASF). The isolated cells were viable for 3-4 h (i.e., over the total time of the experiments), as tested employing morphological parameters of cell damage, reactivity toward trypan blue, and the ability to use succinate in the absence and presence of digitonin. Respiration was activated by addition of sodium ascorbate and tetramethyl-para-phenylenediamine (TMPD) as exogenous reductants, in order to single out the contributions of cytochrome c and cytochrome c oxidase among the complexes of the mitochondrial respiratory chain. TMPD was shown to be freely permeable across cytoplasmic and mitochondrial membranes, with a measured KD = 0.9 mM. The use of singular value decomposition analysis coupled to PASF acquisition proved very powerful in resolving statically and kinetically, in the millisecond time region, the spectral contributions of the cytochromes. Spectral analysis was improved by adding carbon monoxide at concentrations which did not affect cytochrome c oxidase activity, but kept myoglobin fully saturated (and thus uninfluential to absorbance changes).