Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron–electron resonance

Abstract

In oxidative phosphorylation, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. Complex I contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters, in several subunits, to transfer the electrons to quinone. Understanding coupled electron transfer in complex I requires a detailed knowledge of the properties of individual clusters and of the cluster ensemble, and so it requires the correlation of spectroscopic and structural data: This has proved a challenging task. EPR studies on complex I from Bos taurus have established that EPR signals N1b, N2 and N3 arise, respectively, from the 2Fe cluster in the 75 kDa subunit, and from 4Fe clusters in the PSST and 51 kDa subunits (positions 2, 7, and 1 along the seven-cluster chain extending from the flavin). The other clusters have either evaded detection or definitive signal assignments have not been established. Here, we combine double electron-electron resonance (DEER) spectroscopy on B. taurus complex I with the structure of the hydrophilic domain of Thermus thermophilus complex I. By considering the magnetic moments of the clusters and the orientation selectivity of the DEER experiment explicitly, signal N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3). The implications of our assignment for the mechanisms of electron transfer and energy transduction by complex I are discussed.C omplex I (NADH:quinone oxidoreductase) is an essential respiratory enzyme. It is an entry point to the electrontransport chains of many aerobic organisms, and its dysfunction is linked to numerous human diseases by mitochondrial DNA mutations, decreased respiratory capacity and increased oxidative stress (1). Complex I is a complicated, membrane-bound enzyme that couples NADH oxidation and quinone reduction to proton translocation across an energy-transducing membrane. It comprises a membrane extrinsic (hydrophilic) domain, and a membrane intrinsic (hydrophobic) domain. A chain of iron-sulfur (FeS) clusters in the hydrophilic domain is essential in transferring electrons from the flavin mononucleotide at the site of NADH oxidation to the hydrophobic site of quinone reduction. The mechanism by which electron transfer drives proton translocation is not known. The 3.3 Å structure of the hydrophilic domain of Thermus thermophilus complex I, the only high resolution structure available, reveals how the FeS clusters and the flavin are arranged (2). Seven clusters form an extensive chain from the flavin to the quinone binding site; the eighth cluster, of unknown function, is on the opposite side of the flavin (see Fig. 1). A ninth cluster, more than 20 Å from the cluster chain, occurs in only a few species, including T. thermophilus and Escherichia coli. Current knowledge of the properties of the clusters, most obviously their reduction potentials, has been derived ...

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

“…The second possibility would be in agreement with the cluster assignment proposed in the preceding paper. In that case, the basis for the interpretation of the DEER data (Roessler et al 2010) would collapse. Also relevant to this point is that spin-spin interaction of a [4Fe-4S] cluster with a flavin radical has not yet been confirmed for Hirst's NADH dehydrogenase which lacks FMN-a.…”

“…Significance of double electron-electron resonance (DEER) spectroscopy of Hirst's NADH dehydrogenase Roessler et al (Roessler et al 2010) have attempted to assign cluster-cluster interactions observed by double electron-electron resonance (DEER) spectroscopy of the reduced enzyme prepared in their laboratory (Sharpley et al 2006). As a basic reference frame the T. thermophilus structure was used.…”

Multi-haem cytochromes inShewanella oneidensisMR-1: structures, functions and opportunities

“…viridis [92]. (c) Free energy landscape for the FeS cluster ET chain in bovine mitochondrial complex I [93,94], including estimates for the three low-potential clusters (below 20.4 V) [88]. (d ) Visualization of the free energy landscape within the molecular structure of MtrF: haems are coloured according to their redox potential, with lighter colours corresponding to lower redox potentials and thus a higher position in the free energy landscape.…”

Hole hopping through tyrosine/tryptophan chains protects proteins from oxidative damage

“…Electron (or hole) hopping in proteins can involve a series of redox cofactors as in Complex I (NADH dehydrogenase) of the mitochondrial respiratory chain (15)(16)(17). The side chains of select amino acids also can serve as waystations for redox hopping reactions, with Cys, Met, Trp, and Tyr being the most likely candidates (18).…”