Functional Significance of Conserved Histidines and Arginines in the 49-kDa Subunit of Mitochondrial Complex I

Ljuban Grgic,Klaus Zwicker,Noushin Kashani-Poor,Stefan Kerscher,Ulrich Brandt

doi:10.1074/jbc.m313180200

Ljuban Grgic, Klaus Zwicker + Show 3 more

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https://doi.org/10.1074/jbc.m313180200

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Abstract

We have studied the ubiquinone-reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica by a series of point mutations replacing conserved histidines and arginines in the 49-kDa subunit. Our results show that histidine 226 and arginine 141 probably do not ligate iron-sulfur cluster N2 but that exchanging these residues specifically influences the properties of this redox center. Histidines 91 and 95 were found to be essential for ubiquinone reductase activity of complex I. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilized complex I. These results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and its ancestor, the large subunit of water-soluble [NiFe] hydrogenases. In several mutations of histidine 226, arginine 141, and arginine 466 the characteristic EPR signatures of iron-sulfur cluster N2 became undetectable, but specific, inhibitor-sensitive ubiquinone reductase activity was only moderately reduced. As we could not find spectroscopic indications for a modified cluster N2, we concluded that these complex I mutants were lacking most of this redox center but were still capable of catalyzing inhibitor-resistant ubiquinone reduction at near normal rates. We discuss that this at first surprising scenario may be explained by electron transfer theory; after removal of a single redox center in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. Our results question some of the central mechanistic functions that have been put forward for iron-sulfur cluster N2.

Highlights

IntroductionA fourth cysteine is found immediately adjacent to the first of these three conserved cysteines in all known PSST sequences; for steric reasons, it has been generally considered unlikely that the resulting putative Cys-Cys-Xn-CysXn-Cys motif ligates iron-sulfur cluster N2
The respiratory chain of the inner membrane of mammalian mitochondria consists of four enzyme complexes and creates the transmembrane protonmotive force used by ATP synthase to generate ATP
Based on a site-directed mutagenesis study we have proposed that the ubiquinone-reducing catalytic core of complex I has evolved from the active center of [NiFe] hydrogenases and resides at the interface between the 49-kDa and the PSST subunits [4, 5]

Summary

Introduction

A fourth cysteine is found immediately adjacent to the first of these three conserved cysteines in all known PSST sequences; for steric reasons, it has been generally considered unlikely that the resulting putative Cys-Cys-Xn-CysXn-Cys motif ligates iron-sulfur cluster N2 This option has been put forward recently by Flemming et al [12]. As cluster N2 is predicted to reside immediately at the interface between the two subunits, we have suggested that the missing fourth ligand may be provided by the adjacent 49-kDa subunit [4] To explore this possibility and to gain further insight into structure-function relationships within the ubiquinone-reducing catalytic core of complex I, we have analyzed mutations of highly conserved histidines and arginines in the 49-kDa subunit of complex I (Fig. 1). The residues chosen for mutagenesis were predicted to be in the vicinity (within about 15 Å) of iron-sulfur cluster N2 based on the previously demonstrated conservation of the [NiFe] hydrogenase structural fold [4]

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