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.

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 O 2 /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 O 2 /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 a 3 -Cu B binuclear center. The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a 3 . Reversal of inhibition in the presence of O 2 does not involve a direct reaction of O 2 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 O 2 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 a 3 -Cu B center.

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].

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.

The kinetics and stoichiometry of the redox-linked protonation of the soluble Paracoccus denitrificans cytochrome c oxidase were investigated at pH = 7.2-7.5 by multiwavelength stopped-flow spectroscopy, using the pH indicator phenol red. We compared the wild-type enzyme with the K354M and the D124N subunit I mutants, in which the K- and D-proton-conducting pathways are impaired, respectively. Upon anaerobic reduction by Ru-II hexamine, the wild-type enzyme binds 3.3 +/- 0.6 H(+)/aa(3), i.e., approximately 1 H(+) in excess over beef heart oxidase under similar conditions and the D124N mutant 3.2 +/- 0.5 H(+)/aa(3). In contrast, in the K354M mutant, in which the reduction of heme a(3)-Cu(B) is severely impaired, approximately 0.8 H(+) is promptly bound synchronously with the reduction of heme a, followed by a much slower protonation associated with the retarded reduction of the heme a(3)-Cu(B) site. These results indicate that complete reduction of heme a (and Cu(A)) is coupled to the uptake of approximately 0.8 H(+), which is independent of both H(+)-pathways, whereas the subsequent reduction of the heme a(3)-Cu(B) site is associated with the uptake of approximately 2.5 H(+) transferred (at least partially) through the K-pathway. On the basis of these results, the possible involvement of the D-pathway in the redox-linked protonation of cytochrome c oxidase is discussed.

The aa 3 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 215Ϯ 20 mV and 415 Ϯ20 mV at pH 5.4, showing redox-Bohr effect, and a heme a 3 -Cu B center with an affinity for carbon monoxide (K a ϭ 5.7ϫ104 M Ϫ1 at 35°C) much lower than that reported for the mammalian enzyme (K a ϭ 4ϫ10 6 M Ϫ1 at 20°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 a 3 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.

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa 3 ) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a 3 reduction, 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 a 3 reduction in the K354M mutant but only a ϳ5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O 2 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 a 3 reduction is ϳ100-fold faster than the extremely low turnover rate, we conclude that, contrary to O 2 , NO can react not only with the twoelectron 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.

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