Title

Ubiquinone Pair in the Q0 Site Central to the Primary Energy Conversion Reactions of Cytochrome bc1 Complex

Document Type

Article

Publication Date

1-1-1995

Abstract

The mechanistic heart of the ubihydroquinone-cytochrome c oxidoreductase (cyt bc1 complex) is the catalytic oxidation of ubihydroquinone (QH2) at the Q0 site. QH2 oxidation is initiated by ferri-cyt c, mediated by the cyt c1 and [2Fe-2S] cluster of the cytochrome bc1 complex. QH2 oxidation in turn drives transmembrane electronic charge separation through two b-type hemes to another ubiquinone (Q) at the Qi site. In earlier studies, residues F144 and G158 of the b-heme containing polypeptide of the Rhodobacter capsulatus cyt bc1 complex were shown to be influential in Q0 site function. In the present study, F144 and G158 have each been singly substituted by neutral residues and the dissociation constants measured for both Q and QH2 at each of the strong and weak binding Q0 site domains (Qos and Qow). Various substitutions at F144 or G158 were found to weaken the affinities for Q and QH2 at both the Qos and Qow domains variably from zero to beyond 103-fold. This produced a family of Q0 sites with Qos and Qow domain occupancies ranging from nearly full to nearly empty at the prevailing ~3 x10-2 M concentration of the membrane ubiquinone pool (Qpooi). In each mutant, the affinity of the Qos domain remained typically 10-20-fold higher than that of the Qow domain, as is found for wild type, thereby indicating that the single mutations caused comparable extents of the weakening at each domain. Moreover, the substitutions were found to cause similar decreases of the affinities of both Q and QH2 in each domain, thereby maintaining the Q/QH2 redox midpoint potentials (Em7) of the Q0 site at values similar to that of the wild type. Measurement of the yield and rate of QH2 oxidation generated by single turnover flashes in the family of mutants suggests that the Qos and Qow domains serve different roles for the catalytic process. The yield of the QH2 oxidation correlates linearly with Qos domain occupancy (QH2 or Q), suggesting that the Qos domain exchanges Q or QH2 with the Qpooi at a rate which is much slower than the time scale of turnover. On the other hand, the rate constants of the first QH2 oxidation, ranging in the mutants from 1620 to <5 s-1, correlate with the kd values of QH2 and Q at the Qow domain in a simple kinetic model in which the Qow domain exchanges Q or QH2 with the Qpooi at a rate which is much faster than the time scale of turnover as constrained by the kcat (approximately 1700 s-1). The second QH2 oxidation at the Q0 site (required for completion of the catalytic turnover of the cyt bc1 complex) proceeds maximally at 350 s-1 in the wild type, and the yield and rate are affected by the single substitutions at F144 and G158 in parallel to those of the first QH2 oxidation. A plausible mechanism is presented in which the two ubiquinones of the Q0 site cooperate in the primary steps of the catalytic action of the cyt bc1 complex. Key features of the mechanism are as follows: (1) The formation of ubisemiquinone in both the Qos and Qow domains is highly unfavorable. This keeps the steady-state concentration of the reactive semiquinone to vanishingly low levels, and hence diminishes wasteful side reactions. (2) The Qos and Qow domains provide a conduit for the rapid movement of semiquinone away from the oxidizing side (the [2Fe-2S] cluster, cyt c1 and cyt C2) to reduce the cyt bL. This process confers the directional specificity of the reaction, and minimizes the lifetime of semiquinone and wasteful side reactions. (3) A linear arrangement of the ubiquinones in the Qos and Qow domains allows the position of the cyt bL to be at a maximum distance from the [2Fe-2S] cluster and thus stabilizes ferro-cyt bL with respect to the wasteful back-reaction from ferro-cyt bL to reoxidized [2Fe-2S] cluster. This strongly favors the physiologically useful electron transfer from ferro-cyt bL to ferri-cyt bH and the Q in the Qi site. © 1995, American Chemical Society. All rights reserved.

Publication Source (Journal or Book title)

Biochemistry

First Page

15979

Last Page

15996

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