Until recently, two processes were thought to account for all energy conservation in biological systems: substrate-level phosphorylation and electron transport phosphorylation. Recently, a third bioenergetic process was discovered, flavin-based electron bifurcation,1 in which an energetically favorable redox event is coupled with an energetically unfavorable one to conserve energy. Mechanistically, bifurcation requires a two-electron cofactor with a high energy, one-electron reduced state. This leads to the cofactor serving as a site of transduction between two-electron and one-electron chemistry, and the highly reducing one-electron intermediate state is able to perform the energetically unfavorable reaction. In most electron bifurcating enzymes, a flavin is hypothesized to fulfill this role. However, the means by which the protein scaffold tunes the reduction potentials of the flavin to achieve this activity and prevent short-circuiting reactions is currently unknown. To address these questions, we are using protein film electrochemistry, a technique in which a redox enzyme is immobilized on an electrode surface and can exchange electrons directly with the solid substrate, to characterize the catalytic activity of an NADH-dependent reduced ferredoxin:NADP+ oxidoreductase from Pyrococcus furiosus (PfNfnI). This bifurcating enzyme reversibly couples the exergonic reduction of NADP+ by reduced ferredoxin with the endergonic reduction of NADP+ by NADH. We demonstrate that PfNfnI can be functionally immobilized on graphite electrodes and the dependence of catalytic activity on parameters such a substrate identity, electrical potential, and pH will be presented. A mechanism consistent with this work and complementary structural and spectroscopic studies will be presented. References 1. Herrmann, G.; Jayamani, E.; Mai, G.; Buckel, W., J. Bacteriol. 2008, 190 (3), 784-791; Thauer, R.; Kaster, A.; Seedorf, H.; Buckel, W.; Hedderich, R., Nature Rev. Microbiol. 2008, 6 (8), 579-591.

Electrochemical determination of the reactivity of the bifurcating enzyme NADH-dependent reduced ferredoxin:NADP+ oxidoreductase

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.