A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.     

Reaction of dioxygen with cytochrome c oxidase reduced to different degrees: indications of a transient dioxygen complex with copper-B.

The reactions of the fully reduced, three-electron-reduced, and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, starting from the CO complexes, at 445 and 830 nm at pH 7.4 and 25 degrees C. With the fully reduced and the three-electron-reduced enzyme, four kinetic phases with rate constants in the range from 1 x 10(5) to 10(3) s-1 can be observed. The initial fast phase is associated with an absorbance increase at 830 nm. This is followed by an absorbance decrease (2.8 x 10(4) s-1), the amplitude of which increases with the degree of reduction of the oxidase. The third phase (6 x 10(3) s-1) displays the largest absorbance change at both wavelengths in the fully reduced enzyme and is not seen in the mixed-valence oxidase at 830 nm; a change with opposite sign but with a similar rate constant is found at 445 nm in this enzyme form. The slowest phase (10(3) s-1) is also largest in the fully reduced oxidase and not seen in the mixed-valence enzyme. It is suggested that O2 initially binds to reduced CuB and is then transferred to cytochrome a3 before electron transfer from cytochrome a/CuA takes place. The fast oxidation of cytochrome a seen with the fully reduced enzyme is suggested not to occur during natural turnover. A reaction cycle for the complete turnover of the enzyme is presented. In this cycle, the oxidase oscillates between electron input and output states of the proton pump, characterized by cytochrome a having a high and a low reduction potential, respectively.     

Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy

Photobiomodulation by light in the red to near infrared range (630-1000 nm) using low energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing, improve recovery from ischemic injury in the heart and attenuate degeneration in the injured optic nerve. Recent evidence indicates that the therapeutic effects of red to near infrared light result, in part, from intracellular signaling mechanisms triggered by the interaction of NIR light with the mitochondrial photoacceptor molecule cytochrome c oxidase. We have demonstrated that NIR-LED photo-irradiation increases the production of cytochrome oxidase in cultured primary neurons and reverses the reduction of cytochrome oxidase activity produced by metabolic inhibitors. We have also shown that NIR-LED treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. Photobiomodulation improves wound healing in genetically diabetic mice by upregulating genes important in the promotion of wound healing. More recent studies have provided evidence for the therapeutic benefit of NIR-LED treatment in the survival and functional recovery of the retina and optic nerve in vivo after acute injury by the mitochondrial toxin, formic acid generated in the course of methanol intoxication. Gene discovery studies conducted using microarray technology documented a significant upregulation of gene expression in pathways involved in mitochondrial energy production and antioxidant cellular protection. These findings provide a link between the actions of red to near infrared light on mitochondrial oxidative metabolism in vitro and cell injury in vivo. Based on these findings and the strong evidence that mitochondrial dysfunction is involved in the pathogenesis of numerous diseases processes, we propose that NIR-LED photobiomodulation represents an innovative and non-invasive therapeutic approach for the treatment of tissue injury and disease processes in which mitochondrial dysfunction is postulated to play a role including diabetic retinopathy, age-related macular degeneration, Leber's hereditary optic neuropathy and Parkinson's disease.     

Cytochrome c oxidase of Pseudomonas AM 1: purification, and molecular and enzymatic properties

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Pseudomonas AM 1 to an electrophoretically homogeneous state and some of its properties were studied. The oxidase showed absorption peaks at 428 and 598 nm in the oxidized form, and at 442 and 604 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 602 nm. The enzyme molecule was composed of two kinds of subunits with molecular weights of 50,000 and 30,000 and it contained equimolar amounts of heme a and copper atom. The enzyme rapidly oxidized Candida krusei and horse ferrocytochromes c as well as Pseudomonas AM 1 ferrocytochrome c. The reactions catalyzed by the enzyme were strongly inhibited by KCN.     

Differential Effects of 670 and 830 nm Red near Infrared Irradiation Therapy: A Comparative Study of Optic Nerve Injury,Retinal Degeneration,Traumatic Brain and Spinal Cord Injury

Red/near-infrared irradiation therapy (R/NIR-IT) delivered by laser or light-emitting diode (LED) has improved functional outcomes in a range of CNS injuries. However, translation of R/NIR-IT to the clinic for treatment of neurotrauma has been hampered by lack of comparative information regarding the degree of penetration of the delivered irradiation to the injury site and the optimal treatment parameters for different CNS injuries. We compared the treatment efficacy of R/NIR-IT at 670 nm and 830 nm, provided by narrow-band LED arrays adjusted to produce equal irradiance, in four in vivo rat models of CNS injury: partial optic nerve transection, light-induced retinal degeneration, traumatic brain injury (TBI) and spinal cord injury (SCI). The number of photons of 670 nm or 830 nm light reaching the SCI injury site was 6.6% and 11.3% of emitted light respectively. Treatment of rats with 670 nm R/NIR-IT following partial optic nerve transection significantly increased the number of visual responses at 7 days after injury (P≤0.05); 830 nm R/NIR-IT was partially effective. 670 nm R/NIR-IT also significantly reduced reactive species and both 670 nm and 830 nm R/NIR-IT reduced hydroxynonenal immunoreactivity (P≤0.05) in this model. Pre-treatment of light-induced retinal degeneration with 670 nm R/NIR-IT significantly reduced the number of Tunel+ cells and 8-hydroxyguanosine immunoreactivity (P≤0.05); outcomes in 830 nm R/NIR-IT treated animals were not significantly different to controls. Treatment of fluid-percussion TBI with 670 nm or 830 nm R/NIR-IT did not result in improvements in motor or sensory function or lesion size at 7 days (P>0.05). Similarly, treatment of contusive SCI with 670 nm or 830 nm R/NIR-IT did not result in significant improvements in functional recovery or reduced cyst size at 28 days (P>0.05). Outcomes from this comparative study indicate that it will be necessary to optimise delivery devices, wavelength, intensity and duration of R/NIR-IT individually for different CNS injury types.     

Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1

Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.     

Studies on partially reduced mammalian cytochrome oxidase reactions with ferrocytochrome c.

The kinetics of the electron-transfer process which occurs between ferrocytochrome c and partially reduced mammalian cytochrome oxidase were studied by the rapid spectrophotometric techniques of stopped flow and temperature jump. Stopped-flow experiments showed initial very fast extinction changes at 605 nm and at 563 nm, indicating the simultaneous reduction of cytochrome a and oxidation of ferrocytochrome c. During this 'burst' phase, say the first 50 ms after mixing, it was invariably found that more cytochrome c had been oxidized than cytochrome a had been reduced. This discrepancy in electron equivalents may be accounted for by the rapid reduction of another redox site in the enzyme, possibly that associated with the extinction changes observed at 830 nm. During the incubation period in which the partially reduced oxidase was prepared, the rate of reduction of cytochrome a by ferrocytochrome c, at constant reactant concentrations, decreased with time. Temperature-jump experiments showed the presence of two relaxation processes. The faster of the two phases was assigned to the electron-transfer reaction between cytochrome c and cytochrome a. A study of the concentration-dependence of the reciprocal relaxation time for this phase yielded a rate constant of 9 X 10(6)M-1-s-1 for the electron transfer from cytochrome c to cytochrome a, and a value of 8.5 X 10(6)M-1-s-1 for the reverse reaction. The equilibrium constant for the electron-transfer reaction is therefore close to unity. The slower phase has been interpreted as signalling the transfer of electrons between cytochrome a and another redox site within the oxidase molecule.     

Cytochrome aa3 from the aerobic photoheterotroph Erythrobacter longus: purification, and enzymatic and molecular features

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Erythrobacter longus to homogeneity as judged by polyacrylamide gel electrophoresis, and some of its properties were studied. The spectral properties of the oxidase closely resembled those of mitochondrial and other bacterial cytochromes aa3. The enzyme showed absorption peaks at 430 and 598 nm in the oxidized form, and at 444 and 603 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 600 nm. The enzyme oxidized eukaryotic ferrocytochromes C more rapidly than E. longus ferrocytochrome c. The reactions catalyzed by the enzyme were 50% inhibited by 0.7 microM KCN. The enzyme contained 1 g atom of copper and 1 g atom of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two identical subunits, each with a molecular weight of 43,000.     

The binding of carbon monoxide to cytochrome c oxidase.

The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.     

The respiratory responses to cyanide of a cyanide-resistant Klebsiella oxytoca bacterial strain

The respiratory system of a cyanide-resistant Klebsiella oxytoca was analyzed by monitoring the changes in the cytochrome contents in response to various inhibitors in the presence of various concentrations of cyanide. The cells grown in the medium without cyanide (KCN) have two terminal oxidases, cytochrome d (Ki = 10(-5) M KCN) and o (Ki = 10(-3) M KCN). When cells were grown on medium with 1 mM KCN, the expression of both b-type cytochrome and cytochrome d in the plasma membranes of the cell decreased by more than 50%, while cytochrome o increased by 70%, as compared with the cells grown in the absence of KCN. Two terminal oxidases with Ki values of about 10(-3) M and 1.7 x 10(-2) M KCN were observed in the plasma membrane fractions of the cells growing on KCN enriched medium. 2-n-Heptyl-4-hydroxyquinoline-N-oxide markedly inhibited the oxidation of NADH by the plasma membranes from the cells grown in the medium without KCN, but not in those plasma membranes from KCN-grown cells. The NADH oxidases in plasma membranes of K. oxytoca grown with and without KCN were equally sensitive to UV irradiation. Adding freshly isolated quinone to the UV-damaged plasma membranes restored the NADH oxidase activity from both types of plasma membranes. From these results, we propose the presence of a non-heme type of terminal oxidase to account for the KCN resistance in K. oxytoca.     

Titration and steady-state behaviour of the 830 nm chromophore in cytochrome c oxidase.

Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN'N'-tetramethyl-p-phenylenediamine initially introduces two reducing equivalents per mol of cytochrome aa3. The first equivalent reduces the cytochrome a haem iron; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (e.p.r.-detectable copper) but is probably required for reduction of the e.p.r.-undetectable copper. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with ab increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. It is concluded that the e.p.r.-undetectable copper (CuD) is reduced early in the reaction sequence, whereas the detectable copper (CUD) is reduced late. The latter species is probably that responsible for reduction of the cytochrome a3 haem. The magnetic association between undetectable copper and the a3 haem may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Cyanide binding to bovine heart cytochrome c oxidase depleted of subunit III by treatment with lauryl maltoside

Subunit III was removed from beef heart cytochrome oxidase by incubation of the isolated enzyme at 25 degrees C for 24 h in lauryl maltoside buffer at a detergent to protein ratio of 10:1 (w:w). During the course of the incubation, the reaction of the enzyme with cyanide was followed by spectrophotometry in the Soret region. The starting material binds cyanide in a multiexponential process with 70% of the reaction occurring during the slow phase of the reaction at an observed rate of 3.85 X 10(-5) S-1 with 1 mM KCN. More of the enzyme binds cyanide during the fast phase of the reaction at an observed rate of 3.8 X 10(-3) S-1 as subunit III is removed by lauryl maltoside. After 24 h of incubation in lauryl maltoside, the enzyme reacts with cyanide completely in a rapid, single exponential process. When the protein from such an incubation is recovered by cytochrome c affinity chromatography and analyzed for its subunit content, subunit III is absent. The position of the Soret maximum of the oxidized enzyme shifts from its maximum at 418 nm in the starting material to 422 nm in the subunit III-depleted enzyme. The subunit III-depleted enzyme binds cyanide completely in a simple bimolecular reaction with a rate constant of 3.8 M-1 S-1. We discuss this result in terms of the possible structural and functional roles for subunit III in the cytochrome oxidase complex.     

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.     

Study of cytochrome bo function in Vitreoscilla using a cyo(-) knockout mutant

The bacterium, Vitreoscilla, produces a delta mu(Na+) across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na(+) across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo(-)) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo(-) mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo(-) mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild-type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo(-) mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo(-) mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone.     

Electron transfer between cytochrome a and copper A in cytochrome c oxidase: a perturbed equilibrium study

Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by the perturbed equilibrium method. We have prepared a three-electron-reduced, CO-inhibited form of the enzyme in which cytochrome a and copper A are partially reduced and in an intramolecular redox equilibrium. When these samples were irradiated with a nitrogen laser (0.6-ns, 1.0-mJ pulses) to photodissociate the bound CO, changes in absorbance at 598 and 830 nm were observed which were consistent with a fast electron transfer from cytochrome a to copper A. The absorbance changes at 598 nm gave an apparent rate of 17,000 +/- 2000 s-1 (1 sigma), at pH 7.0 and 25.5 degrees C. These changes were not observed in either the CO mixed-valence or the CO-inhibited fully reduced forms of the enzyme. The rate was fastest at about pH 8.0, falling off toward both lower and higher pHs. There was a small but clear temperature dependence. The process was also observed in the cytochrome c-cytochrome c oxidase high-affinity complex. The electron equilibration measured between cytochrome a and copper A is far faster than any rate measured or inferred previously for this process.     

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen.

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen is measured by transient absorption spectroscopy. The oxygen reaction is initiated by photolytic removal of CO from cytochrome oxidase, using a flash-pumped dye laser. The subsequent reaction of the cytochrome c-cytochrome oxidase complex with oxygen is reported at 550, 605, 744, and 830 nm at different cytochrome c:cytochrome oxidase ratios and different oxygen concentrations. In the absence of cytochrome c the time course of the reaction of the oxidase is well described by a triple exponential process at any of the measured wavelengths. The three processes are well resolved at high O2 levels (i.e. greater than 200 microM), where they reach first-order rate limits of 2.4 x 10(4), 7.5 x 10(3), and 650 s-1. When cytochrome c is added the oxidation of cytochrome a and one of the redox active cooper centers (CuA) are interrupted. The maximal effect of cytochrome c on the oxidation of the oxidase occurs at a c:aa3 ratio of 1. Cytochrome c reacts in a biphasic process with rates of up to 7 x 10(3) and 550 s-1 at high oxygen. The fast phase takes up 60% of the process, and this is independent of the cytochrome c:cytochrome oxidase ratio. The results are discussed in the context of a model in which electron entry into cytochrome oxidase from cytochrome c is via CuA, and cytochrome a functions to mediate electron transfer from CuA to the oxygen binding site. The role of CuA as initial electron acceptor in cytochrome c oxidase is related to its physical proximity to cytochrome c is the cytochrome c-cytochrome oxidase complex.     

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

Electron transfer rates and equilibrium within cytochrome c oxidase.

Intramolecular electron transfer (ET) between the CuA center and heme a in bovine cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methyl nicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 830 nm. After the initial reduction phase, the 830 nm absorption was partially restored, corresponding to reoxidation of the CuA center. Concomitantly, the absorption at 445 nm and 605 nm increased, indicating reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration. This demonstrates that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse (heme a --> CuA) process were found to be 13 000 s-1 and 3700 s-1, respectively, at 25 degrees C and pH 7.4. This corresponds to an equilibrium constant of 3.4 under these conditions. Thermodynamic and activation parameters of the ET reactions were determined. The significance of these results, particularly the observed low activation barriers, are discussed within the framework of the known three-dimensional structure, ET pathways and reorganization energies.