Proton processing at the Qo-site of the bc1 complex of Rhodobacter sphaeroides

Abstract

Movement of the iron sulfur protein extrinsic domain (ISP) during catalysis has focused attention on the role of this group as a substrate, and its participation in formation of the enzyme-substrate (ES-) complex at the quinol oxidizing (Qo-) site. Formation of the ES-complex is reflected in the differential binding of quinol when the ISP is oxidized, and in the displacement of the pK of ISPox, both of which show a binding constant of ~14, as determined from kinetic experiments. In the Qo-site reaction, the first electron transfer from quinol to ISPox is rate limiting (~1.5 103 mol/mol bc1/sec), and shows a high activation barrier (~65 kJ/mol). It is likely that the ES-complex has a configuration similar to that of stigmatellin bound at the site, but with quinol forming H-bonds with His-161 of ISPox and Glu-272 of cytochrome (cyt) b. If this is so, then the slow rate is associated with a relatively short electron transfer path (~7 Å). This paradox can be resolved if the reaction is treated as a proton-coupled electron transfer, in which the low probability of transfer of the proton limits that of the electron: Current speculation on the mechanism for transfer of the second electron (from semiquinone to heme bL) centers on a controversy between single- and double-occupancy models. The role of Glu-272 in processing the second proton is critical; this residue rotates through 120o between conformations in stigmatellin and myxothiazol crystals, providing a pathway through which a proton from the H-bond with quinol in the ES-complex could be transferred to a buried water chain, which contacts the carboxylate moiety in the myxothiazol configuration, and could thus deliver a H+ to the external aqueous phase. Since Glu-272 occupies a substantial fraction of the proximal domain of the Qo-site in the stigmatellin configuration (and hence would in the ES-complex), such a role in normal mechanism would likely preclude a double-occupancy in which a second quinone occupied this domain.