Abstract
NADH:quinone oxidoreductase (complex I) couples NADH oxidation and quinone reduction to proton translocation across an energy-transducing membrane. All complexes I contain a flavin to oxidize NADH, seven iron–sulfur clusters to transfer electrons from the flavin to quinone and an eighth cluster (N1a) on the opposite side of the flavin. The role of cluster N1a is unknown, but Escherichia coli complex I has an unusually high-potential cluster N1a and its reduced flavin produces H2O2, not superoxide, suggesting that cluster N1a may affect reactive oxygen species production. In the present study, we combine protein film voltammetry with mutagenesis in overproduced N1a-binding subunits to identify two residues that switch N1a between its high- (E. coli, valine and asparagine) and low- (Bos taurus and Yarrowia lipolytica, proline and methionine) potential forms. The mutations were incorporated into E. coli complex I: cluster N1a could no longer be reduced by NADH, but H2O2 and superoxide production were unaffected. The reverse mutations (that increase the potential by ~0.16 V) were incorporated into Y. lipolytica complex I, but N1a was still not reduced by NADH. We conclude that cluster N1a does not affect reactive oxygen species production by the complex I flavin; it is probably required for enzyme assembly or stability.
Highlights
Complex I (NADH:quinone oxidoreductase) is an essential respiratory enzyme in many organisms
Our first objective was to identify the residues that determine whether N1a is a ‘high-potential’ cluster, as in E. coli, or a ‘lowpotential’ cluster, as in B. taurus and Y. lipolytica (Figure 2)
We focus on the proteins from B. taurus and E. coli, and Y. lipolytica, a yeast model system for mitochondrial complex I that enables mutagenesis in the intact enzyme [40]
Summary
Complex I (NADH:quinone oxidoreductase) is an essential respiratory enzyme in many organisms. In the NADH-reduced V96P variant the intensity of signal N1a is decreased by ∼40 % relative to the wild-type (the spectra in Figure 4 have been normalized to protein concentration so the N1b signal, from a different [2Fe– 2S] cluster, does not change).
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