Iron−Sulfur Cluster N7 of the NADH:Ubiquinone Oxidoreductase (Complex I) Is Essential for Stability but Not Involved in Electron Transfer

Abstract

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN. It catalyzes the electron transfer from NADH to ubiquinone by a chain of redox groups consisting of one FMN and seven iron-sulfur clusters. The function of the additional, nonconserved cluster N7 located on NuoG is not known. It has been speculated that it is not involved in electron transfer, due to its distance of more than 20 A from the electron transfer chain. Dithionite-reduced minus NADH-reduced EPR difference spectra of complex I and of a soluble fragment containing NuoG revealed for the first time the EPR spectrum of N7 in the complex. Individual mutation of the cysteines ligating this cluster to alanine led to a decreased amount of complex I in the membrane without affecting the electron transfer activity. Sucrose gradient centrifugation revealed that the complex from the C230A and C233A mutants decayed in detergent solution while the C237A and C265A mutant complex was stable. Cluster N7 was detectable in the latter mutants but with shifted g-values, indicating a different ligation of N7. Thus, N7 is essential for the stability of the complex but is not involved in electron transfer.

Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

“…-Red-mediated Recombineering-Electrocompetent DH5-␣⌬nuo/pKD46 cells were prepared and electroporated (28). The nptI-sacRB cartridge was amplified from pVO1100 by PCR with the primer pair nuoF::nptI-sacRB (supplemental Table S2).…”

“…Other EPR conditions were as follows: microwave frequency, 9.44 GHz; modulation amplitude, 0.6 mT; time constant: 0.124 s; scan rate: 17.9 mT/min. (28). The reaction of complex I (a) and the E183H F variant (c) was started by adding either NADH (black curve) or NADPH (red curve) as indicated.…”

Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

“…The peripheral arm contains all the prosthetic groups, including one flavin (FMN) at the NADH binding site, and eight Fe-S clusters. Some bacterial versions of Complex I contain an additional Fe-S cluster that is not thought to be part of the main pathway (19). The membrane arm of the mitochondrial enzyme contains all of the seven mitochondria-encoded subunits, and the bacterial enzyme contains homologs of each.…”

Real-time electron transfer in respiratory complex I

“…The other Fe/S clusters seem to constitute an ''electron wire'' from FMN to N2, with the exception of N1a, which curiously appears to be on a ''side path,'' and center N7, which is located far from the electron wire and does not participate in electron transfer ( Fig. 1) (7,8).…”