The High-Spin Heme b L Mutant Exposes Dominant Reaction Leading to the Formation of the Semiquinone Spin-Coupled to the [2Fe-2S]+ Cluster at the Qo Site of Rhodobacter capsulatus Cytochrome bc 1.

Marcin Sarewicz,Małgorzata Wolska,Sebastian Pintscher,Artur Osyczka Artur Osyczka,Łukasz Bujnowicz

doi:10.3389/fchem.2021.658877

Abstract

Cytochrome bc 1 (mitochondrial complex III) catalyzes electron transfer from quinols to cytochrome c and couples this reaction with proton translocation across lipid membrane; thus, it contributes to the generation of protonmotive force used for the synthesis of ATP. The energetic efficiency of the enzyme relies on a bifurcation reaction taking place at the Qo site which upon oxidation of ubiquinol directs one electron to the Rieske 2Fe2S cluster and the other to heme b L. The molecular mechanism of this reaction remains unclear. A semiquinone spin-coupled to the reduced 2Fe2S cluster (SQo-2Fe2S) was identified as a state associated with the operation of the Qo site. To get insights into the mechanism of the formation of this state, we first constructed a mutant in which one of the histidine ligands of the iron ion of heme b L Rhodobacter capsulatus cytochrome bc 1 was replaced by asparagine (H198N). This converted the low-spin, low-potential heme into the high-spin, high-potential species which is unable to support enzymatic turnover. We performed a comparative analysis of redox titrations of antimycin-supplemented bacterial photosynthetic membranes containing native enzyme and the mutant. The titrations revealed that H198N failed to generate detectable amounts of SQo-2Fe2S under neither equilibrium (in dark) nor nonequilibrium (in light), whereas the native enzyme generated clearly detectable SQo-2Fe2S in light. This provided further support for the mechanism in which the back electron transfer from heme b L to a ubiquinone bound at the Qo site is mainly responsible for the formation of semiquinone trapped in the SQo-2Fe2S state in R. capusulatus cytochrome bc 1.

Highlights

The cytochrome bc1 complex (Cytbc1) is one of the enzymes involved in energy conversion that takes place in mitochondria and many prokaryotic respiratory chains and anoxygenic photosynthesis (Berry et al, 2000; Crofts, 2004; Sarewicz and Osyczka, 2015)
The enzymatic activity of Cytbc1 in H198N was lost. We used this mutant to test the formation of the semiquinone at the Qo site (SQo)-[2Fe-2S] Rieske iron–sulfur cluster (2Fe2S) state under nonequilibrium conditions when heme bL cannot perform normal electron-relay function between the Qo site and heme bH
This provided an alternative method for verifying the effectiveness of light activation of reaction center (RC) and confirmed their efficient coupling with Cytbc via cytochrome c2 resulting in the oxidation of [2Fe-2S] Rieske iron–sulfur cluster (2Fe2S)

Summary

Introduction

The cytochrome bc complex (Cytbc1) is one of the enzymes involved in energy conversion that takes place in mitochondria and many prokaryotic respiratory chains and anoxygenic photosynthesis (Berry et al, 2000; Crofts, 2004; Sarewicz and Osyczka, 2015). Cytbc is not operating as a typical proton pump that uses special proton channels but it utilizes quinone molecules to transport protons across the lipid bilayers (Al-Attar and de Vries, 2013). This transport is carried out by coupling two opposite redox reactions of quinones at the two catalytic sites that are located within the enzyme structure at the opposite sides of the membrane (Mitchell, 1975; Crofts et al, 1983). The reduction taking place at the Qi site is associated with uptake of two protons from the n side of the membrane This way the protons are transported through the membrane along with pairs of diffusing UQH2/UQ molecules

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Results

Conclusion