ATP-driven proton pumping and passive Fo-mediated proton translocation. Growth on

succinate minimal media indicated no ATP synthesis by the mutants. The observed

effects were shown not to be due to failure of F1Fo ATP synthase to assemble because

treatment with the detergent lauryldimethylamine oxide (LDAO) released Fl from the

prepared membranes and revealed abundant ATP hydrolysis activity. The presence of

assembled F1Fo ATP synthase complexes incorporated with an aarg210 mutant was later

directly confirmed by Dr. James Gardner (167). Substitution with an alanine allowed

passive Fo-mediated proton translocation indicating that the proton channel was intact

and suggested that the aarg210 iS not obligatorily protonated or deprotonated during proton

conduction (168). A second site suppressor mutation, agln252 arg, which partially

compensated for the aarg210 gln mutation, was identified, and suggested to be in close

proximity to each other with residence on the transmembrane helix 5 and 4, respectively,

in the five-transmembrane model (121). The a210 TOSidue is thought to have a direct role

in proton translocation. The orientation of the a subunit' s fourth transmembrane helix

had been determined relative to the orientation of the c subunit' s second transmembrane

co-helix by crosslinking double cysteine mutants (157). Crosslinking data has positioned

a214 in ClOse proximity to c62 and c65, and a211 ClOse to c69 (157). This places the putative

fourth helix of the a subunit in contact with the second helix of the c subunit. Models

have the a210 TOSidue positioned near the center of the fourth helix at a level in the lipid

bilayer very close to the essential casp61 TOSidue (14). Whether a210 iS directly

protonated/deprotonated or controls protonation of the casp61 TOSidue remain unanswered

(169). Insight from a high resolution structure of an intact Fo is greatly desired and

would provide extremely valuable answers to many of the unsolved questions.