Kinetics of electron and proton transfer during O-2 reduction in cytochrome aa(3) from A-ambivalens: an enzyme lacking Glu(I-286)

Acidianus ambivalens is a hyperthermoacidophilic archaeon which grows optim ally at similar to 80 degreesC and pH 2.5. The terminal oxidase of its resp iratory system is a membrane-bound quinol oxidase (cytochrome aa(3)) which belongs to the heme-copper oxidase superfamily. One difference between this quinol oxidase and a majority of the other members of this family is that it lacks the highly-conserved glutamate (Glu(I-286), E. coli ubiquinol oxid ase numbering) which has been shown to play a central role in controlling t he proton transfer during reaction of reduced oxidases with oxygen. In this study we have investigated the dynamics of the reaction of the reduced A. ambivalens quinol oxidase with O-2. With the purified enzyme, two kinetic p hases were observed with rate constants of 1.8 . 10(4) s(-1) (at 1 mM O-2, pH 7.8) and 3.7 x 10(3) s(-1), respectively. The first phase is attributed to binding of O-2 to heme a(3) and oxidation of both hemes forming the 'per oxy' intermediate. The second phase was associated with proton uptake from solution and it is attributed to formation of the 'oxo-ferryl' state, the f inal state in the absence of quinol. In the presence of bound caldariella q uinol (QH(2)), heme a was re-reduced by QH(2) with a rate of 670 s(-1), fol lowed by transfer of the fourth electron to the binuclear center with a rat e of 50 s(-1). Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center. Mo reover, the overall electron and proton-transfer kinetics in the A. ambival ens quinol oxidase are the same as those in the E. coli ubiquinol oxidase, which indicates that in the A. ambivalens enzyme a different pathway is use d for proton transfer to the binuclear center and/or other protonatable gro ups in an equivalent pathway are involved. Potential candidates in that pat hway are two glutamates at positions (I-80) and (I-83) in the A. ambivalens enzyme (corresponding to Met(I-116) and Val(I-119), respectively, in E. co li cytochrome bos). (C) 2001 Elsevier Science B.V. All rights reserved.