Mitochondrial disease-related mutations at the cytochrome b-iron–sulfur protein (ISP) interface: Molecular effects on the large-scale motion of ISP and superoxide generation studied in Rhodobacter capsulatus cytochrome bc1

Robert Ekiert,Arkadiusz Borek,Patryk Kuleta,Justyna Czernek,Artur Osyczka

doi:10.1016/j.bbabio.2016.03.022

Abstract

One of the important elements of operation of cytochrome bc1 (mitochondrial respiratory complex III) is a large scale movement of the head domain of iron–sulfur protein (ISP-HD), which connects the quinol oxidation site (Qo) located within the cytochrome b, with the outermost heme c1 of cytochrome c1. Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations — G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme [Borek et al., J. Biol. Chem. 290 (2015) 23781-23792]. This resulted in the enhanced propensity of the mutant to generate reactive oxygen species (ROS) which was explained on the basis of the model evoking “semireverse” electron transfer from heme bL to quinone. Here we examine another mutation from that group — G332D (G290D in human), finding that it also shifts the equilibrium position of ISP-HD in the same direction, however displays less of the enhancement in ROS production. We provide spectroscopic indication that G332D might affect the electrostatics of interaction between cytochrome b and ISP-HD. This effect, in light of the measured enzymatic activities and electron transfer rates, appears to be less severe than structural distortion caused by proline in G167P mutant. Comparative analysis of the effects of G332D and G167P confirms a general prediction that mutations located at the cytochrome b-ISP-HD interface influence the motion of ISP-HD and indicates that “pushing” ISP-HD away from the Qo site is the most likely outcome of this influence. It can also be predicted that an increase in ROS production associated with the “pushing” effect is quite sensitive to overall severity of this change with more active mutants being generally more protected against elevated ROS.This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi.

Highlights

Cytochrome bc1, a part of electron transport chain (ETC), is involved in building of proton motive force which is utilized to produce ATP [1]
As the anoxygenic growth of R. capsulatus is dependent on the functional electron transport chain, the lower growth rate of bacteria bearing mutated cytochrome bc1 complex suggests impediments in the complex operation
Upon introducing the G332D mutation in the cytochrome b in R. capsulatus we observed that the growth rate of mutated bacteria was dramatically slower than in the wild type (Fig. 2A)

Summary

Introduction

Cytochrome bc (mitochondrial complex III), a part of electron transport chain (ETC), is involved in building of proton motive force which is utilized to produce ATP [1]. This protein is a homodimer, in which each. 1 These authors contributed to this work. Monomer contains from 3 to 11 subunits, depending on species [2]. The catalytic core of this protein consists of only 3 subunits: cytochrome b, cytochrome c1 and iron–sulfur protein (ISP). Its function is related to electron transfer from quinol to cytochrome c and the translocation of protons across the membrane. Communication between these two pools of electron carriers is important for energetic efficiency of ETC [3]

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