Mixed-valence Cytochrome Oxidase Research Articles

Internal Electron Transfer in Cytochrome c Oxidase Is Coupled to the Protonation of a Group Close to the Bimetallic Site

Absorbance changes following CO dissociation by flash photolysis from mixed-valence cytochrome oxidase have been followed in the Soret and alpha regions. Apart from CO dissociation and recombination, three kinetic phases with rate constants in the range 10(5)-10(3) s-1 at pH 7.5 can be resolved in both spectral regions. The slowest one of these phases, which had earlier only been observed in the alpha region, has now been detected in the Soret region by the use of a low CO concentration to slow down the recombination reaction. This phase had been assigned to a structural change, but a kinetic difference spectrum demonstrates that it represents electron transfer from cytochrome a3 to cytochrome a. A kinetic deuterium isotope effect of 2-3 at pH 7.5 suggests that it involves proton transfer as well. The temperature dependence of the reaction gives an Arrhenius activation energy of 42 kJ.mol-1. The reaction is faster at low pH, and the equilibrium is shifted toward cytochrome a as the pH is raised. The rate and equilibrium changes can be described as involving acid-base groups with pKa values of approximately 7.7 and 8.7, respectively. The kinetic results can be simulated on the basis of a model in which one acid-base group interacts with cytochrome a3, so that its pKa drops on oxidation of this center. The group is in proton equilibrium with the solvent via a proton pathway, suggested to be a proton channel. The rate of a shift in the redox equilibrium between the two cytochromes reaches a high limit at low pH, where the channel is saturated with protons.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Uptake and release of protons during the reaction between cytochrome c oxidase and molecular oxygen: a flow-flash investigation

Changes in pH during the reactions of the fully reduced and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, using the pH indicator phenol red. Solubilized enzyme as well as enzyme reconstituted into phospholipid vesicles has been studied. With the solubilized enzyme, a biphasic uptake of one proton from the medium was observed, whereas the reconstituted enzyme gave release of 1.3 protons to the extravesicular medium. It is concluded from these results that a total of two to three protons are taken up during oxidation of the fully reduced enzyme. Kinetic analysis suggests that the proton uptake is initiated by the transfer of the third electron to the oxygen binding site. A reaction scheme that integrates proton transfers and oxygen chemistry is presented.

The effect of pH and temperature on the reaction of fully reduced and mixed-valence cytochrome c oxidase with dioxygen

The reaction of fully reduced and mixed-valence cytochrome oxidase with O2 has been followed in flow-flash experiments, starting from the CO complexes, at 428, 445, 605 and 830 nm between pH 5.8b and 9.0 in the temperature range of 2-40 degrees C. With the fully reduced enzyme, four kinetic phase with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 2.5 x 10(4), 1.0 x 10(4) and 800 s(-1), respectively, are observed. The rates of the three last phases display a very small temperature dependence, corresponding to activation energies in the range 13-54 kJ x mol(-1). The rates of the third and fourth phases decrease at high pH due to the deprotonation of groups with pKa values of 8.3 and 8.8, respectively, but also the second phase appears to have a small pH dependence. In the reaction of the mixed-valence enzyme, three kinetic phases with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 6000 and 150 s(-1), respectively, are observed. The third phase only has a small temperature dependence, corresponding to an activation energy of 20 kJ x mol(-1). No pH dependence could be detected for any phase. Reaction schemes consistent with the experimental observations are presented. The pH dependencies of the rates of the two final phase in the reaction of the fully reduced enzyme are proposed to be related to the involvement of protons in the reduction of a peroxide intermediate. The temperature dependence data suggest that the reorganization energies and driving forces are closely matched in all electron transfer steps with both enzyme forms. It is suggested that the slowest step in the reaction of the mixed-valence enzyme is a conformation change involved in the reaction cycle of cytochrome oxidase as a proton pump.

Electron redistribution in mixed valence cytochrome oxidase following photolysis of carboxy-oxidase.

Absorbance changes at 446 nm in purified cytochrome oxidase following flash photolysis of carboxy-oxidase poised in the mixed valance state at +220 mV show biphasic kinetics. One phase corresponds to CO recombination to ferrous cytochrome a3 with an energy of activation of 9 kcal/mol; the second phase is 3-5 times faster with an energy of activation of 9.15 kcal/mol. Following flash photolysis at approximately -60 degrees C, cytochromes a and c and the 840-nm CuA species are observed to undergo reduction as electrons from ferrous unliganded cytochrome a3 equilibrate with the equipotential redox centers of the oxidase; as CO recombines with ferrous cyochrome a3, these centers are oxidized and the mixed valence carboxy-oxidase is regenerated. Electron redistribution between centers of the oxidase in the forward and reverse directions occurs faster than does the binding of CO.

Low-temperature studies on mixed-valence cytochrome oxidase by using magnetic circular dichroism

The spin states of the haem components of mixed-valence cytochrome oxidase were studied at room temperature and at temperature down to 20K by using magnetic circular dichroism. The room-temperature studies show the presence of a low-spin ferrous haem together with a low-spin ferric haem, which we attribute to heams a3 and a respectively. At temperatures below 100K it appears that the CO of the mixed-valence CO complex may be irreversibly photolysed, and that in this case haems a and a3 assume their high-spin states. Thus in this enzyme haem-haem interactions appear possible at temperatures below 100K.