Crystal structure of bacterial cytochrome bc1 in complex with azoxystrobin reveals a conformational switch of the Rieske iron–sulfur protein subunit

Lothar Esser,Fei Zhou,Chang-An Yu,Di Xia

doi:10.1074/jbc.ra119.008381

Abstract

Cytochrome bc1 complexes (cyt bc1), also known as complex III in mitochondria, are components of the cellular respiratory chain and of the photosynthetic apparatus of non-oxygenic photosynthetic bacteria. They catalyze electron transfer (ET) from ubiquinol to cytochrome c and concomitantly translocate protons across the membrane, contributing to the cross-membrane potential essential for a myriad of cellular activities. This ET-coupled proton translocation reaction requires a gating mechanism that ensures bifurcated electron flow. Here, we report the observation of the Rieske iron-sulfur protein (ISP) in a mobile state, as revealed by the crystal structure of cyt bc1 from the photosynthetic bacterium Rhodobacter sphaeroides in complex with the fungicide azoxystrobin. Unlike cyt bc1 inhibitors stigmatellin and famoxadone that immobilize the ISP, azoxystrobin causes the ISP-ED to separate from the cyt b subunit and to remain in a mobile state. Analysis of anomalous scattering signals from the iron-sulfur cluster of the ISP suggests the existence of a trajectory for electron delivery. This work supports and solidifies the hypothesis that the bimodal conformation switch of the ISP provides a gating mechanism for bifurcated ET, which is essential to the Q-cycle mechanism of cyt bc1 function.

Highlights

Cytochrome bc1 complexes, known as complex lar respiratory chain [1]
iron–sulfur protein (ISP)–ED conformation switch is characterized by the change from a fixed to a mobile state and induced by different types of QP site inhibitors
The iron–sulfur protein’s extrinsic domain (ISP–ED) conformation switch was first suggested in a structural study of mitochondrial cyt bc1, in which different inhibitors gave rise to dramatically different peak heights in anomalous difference Fourier maps for the Fe2S2 cluster at its cyt b– binding site [10]

Summary

Results

We previously introduced a double-cysteine mutation in Rsbc to show that the flexibility of the ISP–ED is important for the cyt bc function [23]. Unlike the static head domain of cyt c1, the ISP–ED is mobile and disordered in both halves of the cyt bc dimer, with no clearly defined electron density to model the polypeptide chain (Fig. 3B) Both ISP–EDs are so disordered that the Fe2S2 cluster can only be located with the help of its anomalous signal, which is obtained from the anomalous difference Fourier map using the coefficients F(h,k,l) – F(-h,-k,-l) with a phase of ␾model Ϫ90°. The binding of azoxystrobin allows the ISP–ED to be in a mobile state with no specific positional preference This degree of mobility is observed in mitochondrial cyt bc in the structures Btbc1/azo (PDB code 1SQB) and Ggbc1/azo (PDB code 3L71). Statistics on the quality of diffraction data sets of bc crystals and structural models

Diffraction data

PDB entry

Discussion

Cyt bB

Gallus gallus

Experimental procedures