Using a chimeric respiratory chain and EPR spectroscopy to determine the origin of semiquinone species previously assigned to mitochondrial complex I

John J Wright,Justin G Fedor,Maxie M Roessler,Judy Hirst

doi:10.1186/s12915-020-00768-6

Abstract

BackgroundFor decades, semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis. However, the difficulty in investigating complex I semiquinones, due to the many different enzymes embedded in the inner mitochondrial membrane, has resulted in an ambiguous picture and no consensus.ResultsIn this paper, we re-examine the highly debated origin of semiquinone species in mitochondrial membranes using a novel approach. Our combination of a semi-artificial chimeric respiratory chain with pulse EPR spectroscopy (HYSCORE) has enabled us to conclude, unambiguously and for the first time, that the majority of the semiquinones observed in mitochondrial membranes originate from complex III. We also identify a minor contribution from complex II.ConclusionsWe are unable to attribute any semiquinone signals unambiguously to complex I and, reconciling our observations with much of the previous literature, conclude that they are likely to have been misattributed to it. We note that, for this earlier work, the tools we have relied on here to deconvolute overlapping EPR signals were not available. Proposals for the mechanism of complex I based on the EPR signals of semiquinone species observed in mitochondrial membranes should thus be treated with caution until future work has succeeded in isolating any complex I semiquinone EPR spectroscopic signatures present.

Highlights

Semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis
To prevent the substrate from becoming exhausted and ensure turnover during electron paramagnetic resonance (EPR) sample preparation, alternative oxidase (AOX) was titrated to a low level, at which the flux through the complex I-AOX pathway matched that through the canonical chain (Additional file 1, Figure S1)
In line with previous studies [28, 29], respiratory control ratio (RCR) values of 1.6 to 3.0 were typically observed in the absence of AOX, and substantial rates of NAD+ reduction (reverse electron transfer, Reverse electron transfer (RET), driven by a reduced Q-pool formed by succinate oxidation and a proton-motive force (Δp) formed by ATP hydrolysis) were observed in both cases (Table 1)

Summary

Introduction

Semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis. The key ubiquinone reduction step in catalysis must proceed via a semiquinone (SQ) radical, given that the iron-sulphur (Fe-S) clusters linking the two substrate binding sites are single electron carriers. It is no Wright et al BMC Biology (2020) 18:54 surprise that, over the past four decades, electron paramagnetic resonance (EPR) spectroscopy has been used extensively to investigate the SQ species associated with complex I. Given that different mechanisms have been proposed on the basis of different ubiquinone reduction pathways [15,16,17], a re-investigation of the putative SQs associated with complex I is warranted

Objectives

Methods

Results

Discussion

Conclusion