Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M.
Complex I is an entry point of the electron transport chain and plays a major role in proton gradient generation. Oxidation of NADH, reduction of ubiquinone, and proton translocation are three essential functions of Complex I. Subunits L, M, and N, the three largest subunits in the membrane domain, are homologous to components of multi‐subunit Na+/H+ antiporters, so they are implicated in proton pumping. Based on available evidence it is believed that a redox‐linked conformational change is necessary for proton pumping. According to recent crystal structures, subunit L contains an unusual helix HL extending laterally along the membrane surface from subunit L, past subunit M, and to subunits N and K. In this study, cross‐linking of engineered cysteine residues using disulfide formation and bi‐functional reagents was carried out to detect regions of the proton transporter that have restricted motion. Fourteen cysteine pairs between helix HL and membrane subunits L, M, N and K were cross‐linked and none showed no significant loss of enzyme activity or proton pumping These results suggest that there is not significant movement of the lateral helix. Further cross‐linking studies were done between pairs of cysteine residues within subunit N. The results of those studies suggested possible proton pathways. Grant Funding Source: NIH 1R15GM099014
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