Abstract
We have investigated the kinetics of NAD(+)-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD(+) through successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI's large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD(+); low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting k(red) of 36 ± 1.6 s(-1) and apparent K(d)(NADPH) of 5 ± 1.2 μM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD(+), the system can bifurcate and generate low-potential electrons without NAD(+). When enzyme is reduced by NADPH in the absence of NAD(+) but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD(+) demonstrates that electron bifurcation is an intrinsic property of the bifurcating FAD and is not dependent on the simultaneous presence of NAD(+) and ferredoxin. The tight coupling between NAD(+) and ferredoxin reduction observed under multiple-turnover conditions is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions.