Pia Ädelroth

Factors determining electron-transfer rates in cytochrome c oxidase: investigation of the oxygen reaction in the R. sphaeroides enzyme

We have investigated the kinetics of the single-turnover reaction of fully reduced solubilised cytochrome c oxidase (cytochrome aa3) from Rhodobacter sphaeroides with dioxygen using the flow-flash methodology and compared the results to those obtained with the well-characterised bovine mitochondrial enzyme. The overall reaction sequence was the same in the two enzymes, but the extents and rates of the electron-transfer reactions differed, implying differences in redox potentials, and/or interaction energies between electrons and protons during oxygen reduction. As with the bovine enzyme, the R. sphaeroides enzyme displayed two major kinetic phases of proton uptake with rate constants of approximately 5000 s-1 and approximately 500 s-1 at pH 7.9, concomitant with the peroxy to oxoferryl and oxoferryl to oxidised states. The net number of protons taken up in the R. sphaeroides enzyme was about approximately 1.9, which implies that upon reduction, the enzyme has to pick up approximately 2.1 H+ from the medium. On the basis of the comparison of electron-transfer reactions in the two enzymes, we conclude that the transfer rate of the fourth electron to the binuclear centre is not only determined by the electron-transfer rate from haem a to the binuclear centre, but also by the electron equilibrium between CuA and haem a. In addition, in contrast to the bovine enzyme, where the electron- and proton-transfer rates during oxidation of the fully reduced enzyme by O2 are all faster than the overall turnover rate, in the R. sphaeroides enzyme, the slowest kinetic phase was rate limiting for the overall turnover. Moreover, the comparison of the reactions in the two systems shows that in the R. sphaeroides enzyme, the electrons are more evenly distributed among the redox centres during oxygen reduction. This enables investigations of effects also of minor perturbations on, e.g., the electron-transfer characteristics in mutant enzymes, for which this study forms the basis.

Triplet-state quenching in complexes between Zn-cytochrome c and cytochrome oxidase or its CuA domain

The quenching of the triplet state of Zn-cytochrome c in electrostatic complexes with cytochrome oxidase and its soluble CuA domain has been studied by laser flash photolysis. The triplet state of free Zn-cytochrome c decayed with a rate of about 200 s-1. With the oxidase, biphasic decay with rate constants of 2 x 10(5) and 2 x 10(3) s-1, respectively, was observed. At high ionic strength (I = 0.2) the decay was the same as with free Zn-cytochrome c. The quenching was also eliminated by reduction of the oxidase. The decay rate in the complex with the CuA domain was 4 x 10(4) s-1. The results are interpreted in terms of rapid electron transfer to CuA and a slower one to cytochrome a. No electron transfer products were detected, because the backward reaction is faster than the forward one. This can be explained by the high driving force (1.1 eV) for the forward electron transfer, taking the system into the inverted Marcus region. The distance in the electrostatic complex between cytochrome c and the electron acceptor, presumed to be CuA, is calculated to be 16 A.

EPR studies of wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides: Glu286 is not a bridging ligand in the cytochrome a3-CuB center

Wild‐type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH‐induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging car☐yl ligand in the bimetallic cytochrome a 3‐CuB site, is found also in the bacterial enzyme. Mutation of glutamate‐286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the car☐yl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low‐spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non‐functional.

A FLASH-PHOTOLYSIS STUDY OF THE REACTIONS OF A CAA3-TYPE CYTOCHROME OXIDASE WITH DIOXYGEN AND CARBON MONOXIDE

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 Μs, 30 Μs, 100 Μs, and 1 ms at 445 nm. The initial, 10-Μs absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and CuA are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O2 were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea3 toa in PS3 oxidase compared to the bovine enzyme.