Iron-Sulfur Cluster N2 of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is Located on Subunit NuoB

Abstract

The proton-pumping NADH:ubiquinone oxidoreductase, also called respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to 9 iron-sulfur (Fe/S) clusters participate in the redox reaction. There is discussion that the EPR-detectable Fe/S cluster N2 is involved in proton pumping. However, the assignment of this cluster to a distinct subunit of the complex as well as the number of Fe/S clusters giving rise to the EPR signal are still under debate. Complex I from Escherichia coli consists of 13 polypetides called NuoA to N. Either subunit NuoB or NuoI could harbor Fe/S cluster N2. Whereas NuoB contains a unique motif for the binding of one Fe/S cluster, NuoI contains a typical ferredoxin motif for the binding of two Fe/S clusters. Individual mutation of all four conserved cysteine residues in NuoB resulted in a loss of complex I activity and of the EPR signal of N2 in the cytoplasmic membrane as well as in the isolated complex. Individual mutations of all eight conserved cysteine residues of NuoI revealed a variable phenotype. Whereas cluster N2 was lost in most NuoI mutants, it was still present in the cytoplasmic membranes of the mutants NuoI C63A and NuoI C102A. N2 was also detected in the complex isolated from the mutant NuoI C102A. From this we conclude that the Fe/S cluster N2 is located on subunit NuoB.

Functional Roles of Four Conserved Charged Residues in the Membrane Domain Subunit NuoA of the Proton-translocating NADH-Quinone Oxidoreductase from Escherichia coli

“…Because this length does not allow incorporation of the whole operon into expression vectors, a site-specific mutation is traditionally carried out by complementation of a cassette-inserted gene with a mutated gene in the expression plasmid (designated in trans complementation) (37)(38)(39)(40)(41)(42)(43). However, the in trans complementation procedure presents some problems when applied to a gene cluster.…”

“…Another problem is that the mutated gene is under the control of a promoter in the expression plasmid, which often leads to overexpression of the mutated subunit. In mutation studies of the NDH-1 using the in trans complementation, it has been reported that the enzyme activities were significantly low (ϳ20% of the wild type cells) even when the unmutated gene was used (42,43,45). An alternative method of site-directed mutation is to introduce mutations directly in chromosomal DNA as detailed in this work (designated chromosomal DNA mutation, see Fig.…”

“…An alternative method of site-directed mutation is to introduce mutations directly in chromosomal DNA as detailed in this work (designated chromosomal DNA mutation, see Fig. 1) (29,42). In this procedure, expression of all genes of the operon are regulated by the authentic promoter.…”

Substrate-induced Conformational Change in Bacterial Complex I

“…They may include NuoA, which has been suggested to interact with NuoB (33), or NuoH, which may contain a ubiquinone-binding site (4). It has been suggested that subunit NuoB coordinates iron-sulfur cluster N2 (34), which has a high pH-dependent midpoint potential and is, therefore, proposed to reduce ubiquinone and play a key role in proton translocation (4). The mobility of subunit NuoB, which we observed, would be consistent with such a role and may reflect movements of this subunit during the catalytic cycle.…”

Reevaluating the relationship between EPR spectra and enzyme structure for the iron–sulfur clusters in NADH:quinone oxidoreductase

“…Finally, we are unable to attribute N4 specifically to 4Fe[I]1 or 4Fe[I]2 or to rule out the possibility that both clusters contribute. Point mutants of the Cys residues in E. coli NuoI have been created, but only one variant (C102A, where C102 is a ligand of 4Fe[I]2) was amenable to study by EPR, and the N4 signal remained clearly visible (48).…”