Glutamate 286 in Cytochrome aa₃ from Rhodobacter sphaeroides Is Involved in Proton Uptake during the Reaction of the Fully-Reduced Enzyme with Dioxygen

Abstract

The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

“…3D), followed by a slow (compared with the wildtype CytcO) biphasic decay to the absorbance level of the fully oxidized state with time constants of 30 and 100 ms. Because the absorbance level at 580 nm in the time range around 100 -300 s was higher than the final absorbance level of the E286Q mutant CytcO, which was shown earlier to stop at the P intermediate (27), it is likely that the F intermediate is formed on a Ͻ100-s time scale in the S197D mutant CytcO. However, it is difficult to resolve kinetically its formation, because it is accelerated and displays a rate that is similar to that associated with formation of P R .…”

“…In D, the dashed line represents the final absorbance level measured with the E286Q mutant CytcO (from Ref. 27). E, absorbance changes at 560 nm of the pH dye phenol red during reaction of the fully reduced wild-type and S197D mutant CytcO with O 2 .…”

“…oxoferryl state (F). Formation of F is rate-limited by internal proton transfer from Glu 286 (see below) that is followed by immediate reprotonation of Glu 286 from solution via the D pathway (27)(28)(29)(30). In addition, during the P R 3 F transition, the electron on Cu A equilibrates with heme a, which has been shown to be controlled by the proton uptake from the bulk solution (28,29,31).…”

“…electron transfer from the heme a-Cu A equilibrium to the catalytic site forming the fully oxidized state (O)), proton uptake takes place through the D pathway via Glu 286 (22,30,32). The residue is a highly conserved component of the D pathway, and replacement of Glu 286 by its nonprotonatable analog, Gln, results in impaired proton transfer through the pathway (8,13,27). In both the P M/R 3 F and F 3 O transitions, the residue itself (or together with a cluster of water molecules) is the primary proton donor to the catalytic site, displaying an apparent pK a of 9.4 (30).…”

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

“…The highly exergonic reduction of O 2 to water also drives the translocation of four protons per O 2 molecule across the membrane, from the negative to the positive side. These ''pumped'' protons are presumably all taken up via the D-pathway, which means that this pathway is used for the transfer of six or seven protons per turnover (3,(6)(7)(8)(9)(10)(11). Because both the substrate and pumped protons are taken up through the D-pathway, there has to be a branching-point distributing protons either to the catalytic site or to the acceptor site for pumped protons (''pump site'').…”