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 optimally at approximately 80 degrees C and pH 2.5. The terminal oxidase of its respiratory 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 oxidase numbering) which has been shown to play a central role in controlling the 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 phases were observed with rate constants of 1.8&z.ccirf;10(4) s(-1) (at 1 mM O(2), pH 7.8) and 3. 7x10(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 'peroxy' intermediate. The second phase was associated with proton uptake from solution and it is attributed to formation of the 'oxo-ferryl' state, the final state in the absence of quinol. In the presence of bound caldariella quinol (QH(2)), heme a was re-reduced by QH(2) with a rate of 670 s(-1), followed by transfer of the fourth electron to the binuclear center with a rate of 50 s(-1). Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center. Moreover, the overall electron and proton-transfer kinetics in the A. ambivalens 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 used for proton transfer to the binuclear center and/or other protonatable groups in an equivalent pathway are involved. Potential candidates in that pathway 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. coli cytochrome bo(3)).
Gilderson
G
G
Department of Biochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
Aagaard
A
A
Gomes
C M
CM
Adelroth
P
P
Teixeira
M
M
Brzezinski
P
P
D001105:Archaea / Q000201:enzymology*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D011522:Protons
D011522:Protons; D018698:Glutamic Acid; D003576:Electron Transport Complex IV; D010100:Oxygen