Abstract
Cytochrome c oxidase is the terminal complex of the respiratory chain in mitochondria and some aerobic bacteria and is responsible for most of the O(2) consumption in biology. The key reaction in the catalysis of O(2) reduction is O-O bond scission that requires four electrons and a proton. In our recent work (Gorbikova, E. A., Belevich, I., Wikstrom, M., and Verkhovsky, M. I. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 10733-10737), it was shown that the cross-linked Tyr-280 (Paracoccus denitrificans numbering) provides the proton for O-O bond cleavage. The deprotonated Tyr-280 must be reprotonated later on in the catalytic cycle to serve as a proton donor for the next oxygen reduction event. To find the reaction step at which the cross-linked Tyr-280 becomes reprotonated, all further steps of the catalytic cycle after O-O bond cleavage were followed by infrared spectroscopy. We found that complete reprotonation of the tyrosine is linked to the formation of the one-electron reduced state coupled to reduction of the Cu(B) site.
Highlights
Catalysis by Cytochrome c oxidase (CcO) is initiated by binding of an oxygen molecule to heme a3 when both redox centers of the binuclear active site are reduced
In the case of the mixed valence enzyme, where only heme a3 and CuB are initially reduced but CuA and heme a are oxidized, the fourth electron is extracted from a nearby residue, most likely Tyr-280 [11, 12] that is located in close proximity to the binuclear center, at a distance of ϳ6 Å
We used Fourier transform infrared (FTIR) spectroscopy in kinetic and equilibrium modes, i.e. we monitored the band at 1308 cmϪ1, which was previously assigned to the deprotonated Tyr-280 anion [17], for each step of the catalytic cycle beyond the formation of the PR intermediate
Summary
Catalysis by CcO is initiated by binding of an oxygen molecule to heme a3 when both redox centers of the binuclear active site are reduced. In the case of the mixed valence enzyme, where only heme a3 and CuB are initially reduced but CuA and heme a are oxidized, the fourth electron is extracted from a nearby residue, most likely Tyr-280 [11, 12] that is located in close proximity to the binuclear center, at a distance of ϳ6 Å. This state of the binuclear center has been called PM. Tel.: 358-9-19158005; Fax: FRCO, fully reduced CO-inhibited form of CcO; O, fully oxidized intermediate; P, peroxy intermediate; PM, peroxy intermediate formed from the mixed valence ϭ two-electron reduced enzyme; PR, peroxy intermediate formed from enzyme in the fully reduced state; R, fully reduced binuclear center; ATR, attenuated total reflectance; FTIR, Fourier transform infrared
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