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Partial Steps of Charge Translocation in the Nonpumping N139L Mutant of Rhodobacter sphaeroides Cytochrome c Oxidase with a Blocked D-Channel

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journal contribution
posted on 13.04.2010, 00:00 by Sergey A. Siletsky, Jiapeng Zhu, Robert B. Gennis, Alexander A. Konstantinov
The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in an ∼15-fold decrease in the turnover number and a loss of proton pumping. Time-resolved absorption and electrometric assays of the FO transition in the N139L mutant oxidase result in three major findings. (1) Oxidation of the reduced enzyme by O2 shows ∼200-fold inhibition of the FO step (k ∼ 2 s−1 at pH 8) which is not compatible with enzyme turnover (∼30 s−1). Presumably, an abnormal intermediate Fdeprotonated is formed under these conditions, one proton-deficient relative to a normal F state. In contrast, the FO transition in N139L oxidase induced by single-electron photoreduction of intermediate F, generated by reaction of the oxidized enzyme with H2O2, decelerates to an extent compatible with enzyme turnover. (2) In the N139L mutant, the protonic phase of Δψ generation coupled to the flash-induced FO transition greatly decreases in rate and magnitude and can be assigned to the movement of a proton from E286 to the binuclear site, required for reduction of heme a3 from the Fe4+O2− state to the Fe3+−OH state. Electrogenic reprotonation of E286 from the inner aqueous phase is missing from the FO step in the mutant. (3) In the N139L mutant, the KCN-insensitive rapid electrogenic phase may be composed of two components with lifetimes of ∼10 and ∼40 μs and a magnitude ratio of ∼3:2. The 10 μs phase matches vectorial electron transfer from CuA to heme a, whereas the 40 μs component is assigned to intraprotein proton displacement across ∼20% of the membrane dielectric thickness. This proton displacement might be triggered by rotation of the charged K362 side chain coupled to heme a reduction. The two components of the rapid electrogenic phase have been resolved subsequently with other D-channel mutants as well as with cyanide-inhibited wild-type oxidase. The finding helps to reconcile the unusually high relative contribution of the microsecond electrogenic phase in the bacterial enzyme (∼30%) with the net electrogenicity of the FO transition coupled to transmembrane transfer of two charges per electron.