Investigating the function of [2Fe–2S] cluster N1a, the off-pathway cluster in complex I, by manipulating its reduction potential

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.

The Fumarate Reductase of Bacteroides thetaiotaomicron , unlike That of Escherichia coli , Is Configured so that It Does Not Generate Reactive Oxygen Species

“…It was hypothesized that during turnover, the extra cluster might momentarily sequester an electron from the flavin, minimizing its flavosemiquinone content and thereby diminishing ROS production (51, 52). However, conversion of the N1a cluster to a nonreducible low-potential center did not alter the autoxidation rate of the isolated enzyme (53). Thus, for the moment the disparity in autoxidation behaviors of B. thetaiotaomicron and E. coli Frd enzymes is a singular demonstration that some redox enzymes can acquire structures that suppress their tendency to contribute to oxidative stress.…”

Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria

“…Thermo-flavin experiments were carried in an ABI 7900HT real-time PCR machine, starting at 20°C for 2 min, then stepping by 1.5°C every 30 s to 80°C [32,33]. Aliquots (20 μl) of 1 mg/ml complex I in 20 mM sodium MOPS (pH 7.5) and 200 mM NaCl (plus additives) were dispensed into a 96-well plate and sealed with optically clear seals; fluorescence intensity data were fit with a Boltzmann sigmoid using Prism.…”

Reduction potential calculations of the Fe–S clusters in Thermus thermophilus respiratory complex I

“…The effects of salt concentration were investigated for a[2Fe‐2S] in the isolated Nqo2 subunit (see Methods) in aqueous solution because experimental data exists for Nqo2 from T. thermophilus , E. coli , and B. taurus at 0.01, 0.1, 0.5, and 2.0 M NaCl . This cluster is at the protein surface in the isolated subunit, so salt effects are expected to be larger.…”

“…However, a[2Fe‐2S] is only reduced by NADH in the intact complex (peripheral and membrane domains) from E. coli and a flavoprotein subcomplex (Nqo1 and Nqo2 subunits containing a[2Fe‐2S], FMN, and 1[4Fe‐4S]) from E. coli and Bos taurus but not in complex I from T. thermophilus or B. taurus . Thus, it may instead serve a structural role such as assembly or stability …”