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

July 1st, 2015 by Zhu, S., Vik, S. B.

Complex I (NADH:ubiquinone oxidoreductase) is a multi-subunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in E. coli, has an unusual lateral helix of about 75 residues, that lies parallel to the membrane surface, and has been proposed to play a mechanical role as a piston during proton translocation (Efremov et al. Nature 2010, 465:441-445.) To test this hypothesis we have introduced eleven pairs of cysteine residues into Complex I: in each pair one is in the lateral helix and the other in a nearby region of subunit N, M or L. The double mutants were treated with Cu ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50%-90%, as judged by immuno-blotting, but in no case did the activity of Complex I decrease by more than 10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue E144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.
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