Binding dynamics at the quinone reduction (Qi) site influence the equilibrium interactions of the iron sulfur protein and hydroquinone oxidation (Qo) site of the cytochrome bc1 complex

摘要

Multiple instances of low potential electron transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc1 complex to varying degrees, ranging from changes in hydroquinone (QH2) oxidation and cyt c1 reduction kinetics, to proteolytic accessibility of the hinge region of the iron-sulfur containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low potential electron transport pathway by Qi site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Qi site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster, and a second affecting the mobility of this subunit are revealed. Remarkably, different inhibitors and mutations at or near the Qi site induce these two effects differently, indicating that the events occurring at the Qi site affect the global structure of the cyt bc1. Furthermore, occupancy of discrete Qi site subdomains differently impede the location of the Fe/S protein at the Qo site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH2 and Q at the Qi site, respectively. Implications of these findings in respect to the Qo–Qi sites communications and to multiple turnovers of the cyt bc1 are discussed.