Significance of [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I

Abstract

NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with proton translocation across the membrane. NADH reduces a noncovalently bound FMN, and the electrons are transported further to the quinone reduction site by a 95 Å long chain of seven iron-sulfur (Fe-S) clusters. Binuclear Fe-S cluster N1a is not part of this long chain but is located within electron transfer distance on the opposite site of FMN. The relevance of N1a to the mechanism of complex I is not known. To elucidate its role, we individually substituted the cysteine residues coordinating N1a of Escherichia coli complex I by alanine and serine residues. The mutations led to a significant loss of the NADH oxidase activity of the mutant membranes, while the amount of the complex was only slightly diminished. N1a could not be detected by electron paramagnetic resonance spectroscopy, and unexpectedly, the content of binuclear cluster N1b located on a neighboring subunit was significantly decreased. Because of the lack of N1a and the partial loss of N1b, the variants did not survive detergent extraction from the mutant membranes. Only the C97A variant retained N1a and was purified by chromatographic steps. The preparation showed a slightly diminished NADH/ferricyanide oxidoreductase activity, while the NADH:decyl-ubiquinone oxidoreductase activity was not affected. N1a of this preparation showed unusual spectroscopic properties indicating a different ligation. We discuss whether N1a is involved in the physiological electron transfer reaction.

Iron‐sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

“…It was attempted to directly observe the effects caused by the deletion of the putative carrier proteins on the Fe/S clusters of complex I by EPR‐spectroscopy. Because of the presence of several membrane‐bound proteins containing tetranuclear Fe/S clusters only the signals of the binuclear clusters N1a and N1b can unequivocally be identified when measuring membranes (Dörner et al ., ). To specifically detect these clusters, difference spectroscopy was applied.…”

“…An additional binuclear cluster on NuoE, N1a, is not part of the chain of clusters but is located in electron transfer distance to FMN. The cluster is strictly conserved and plays not only a role in the stability and the assembly of the complex (Birrell et al ., ; Dörner et al ., ) but also in regulating NADH oxidation (Gnandt et al ., ).…”

Biochemical consequences of two clinically relevant ND-gene mutations in Escherichia coli respiratory complex I

“…SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) was performed according to Schägger 80 with a 10% separating gel and a 3.9% stacking gel. EPR spectra were recorded at 40 and 13 K with a Bruker EMX 6/1 spectrometer operating at X-band 33 . The samples were reduced by an addition of 1.000-fold molar excess NADH and a few grains of dithionite.…”

The two redox states of the human NEET proteins’ [2Fe–2S] clusters

“…Both functionals have been widely used to study [2Fe-2S] and [4Fe-4S] clusters' electronic structure [17,21,26]. 2 The geometrical optimization was carried out using the following convergence criterion: the maximum and RMS force on the nuclei are less than 0.00045 Hartrees/Bohr and 0.00035 Hartrees/Bohr, respectively, and the maximum and RMS nuclei displacement are less than 0.0018 and 0.0012 Å, respectively. The structural determinants of the optimized structures are basically identical and therefore the results are reported only for the mNT protein (PDB ID: 2QH7 [22]).…”

“…Two iron-two sulfur [2Fe-2S] proteins perform electron transfer, serve as oxygen/iron sensors and transcription factors, and perform enzymatic reactions and many other functions across the three kingdoms of life [1,2]. They contain a ferrous and ferric or two ferric ions in their reduced and oxidized forms, respectively [3].…”