Electron Transfer in Nitrogenase

Abstract

Nitrogenase is the only enzyme capable of reducing N2 to NH3. This challenging reaction requires the coordinated transfer of multiple electrons from the reductase, Fe-protein, to the catalytic component, MoFe-protein, in an ATP-dependent fashion. In the last two decades, there have been significant advances in our understanding of how nitrogenase orchestrates electron transfer (ET) from the Fe-protein to the catalytic site of MoFe-protein and how energy from ATP hydrolysis transduces the ET processes. In this review, we summarize these advances, with focus on the structural and thermodynamic redox properties of nitrogenase component proteins and their complexes, as well as on new insights regarding the mechanism of ET reactions during catalysis and how they are coupled to ATP hydrolysis. We also discuss recently developed chemical, photochemical, and electrochemical methods for uncoupling substrate reduction from ATP hydrolysis, which may provide new avenues for studying the catalytic mechanism of nitrogenase.

Natural and Engineered Electron Transfer of Nitrogenase

“…This was indeed observed in a few studies using either NifF or Ti(III) [53,63,64]. Yet in most studies the ATP:e − ratio remained at 2:1, even when NifF was used as the reductant [9,16]. In contrast, DT only reduces the [4Fe-4S] cluster to the [4Fe-4S] 1+ state, and thus transfers only one e − per transient association cycle.…”

“…In addition to DT, other electron mediators employed with the Fe protein include MV (and derivatives), Ti(III) citrate, Eu(II) complexes, and one of the presumed in vivo electron donors, Fld (NifF) [9]. While Ti(III) and Eu(II) complexes are useful due to their low reduction potentials, they are typically prepared as a stock of a single-use reductant.…”

“…Oxidized Fe protein is subsequently reduced by flavodoxin and/or ferredoxin in vivo or commonly dithionite (DT) in vitro, thereby restarting the "Fe protein cycle" [10,11]. The reduction potential (E o ') of the [4Fe-4S] 2+/1+ couple of the Fe protein is approximately −0.30 V vs. the standard hydrogen electrode (SHE, see [12,13]), free of MgATP or MgADP [9,14]. Upon the association of MgATP, the E o ' of [4Fe-4S] 2+/1+ is modulated to −0.43 V vs. SHE, which decreases further to −0.62 V when the Fe protein associates with the MoFe protein making it a more potent electron donor [14,15].…”

“…The FeMo-co, the cofactor at which N 2 is reduced, is coordinated by a Cys and a histidine (His) residue. Each αß half of the MoFe protein repeatedly transiently associates with a 1e − -reduced and ATP-bound Fe protein, during which a single electron is ultimately transferred from the [4Fe-4S] 1+ cluster to the FeMo-co via the P cluster [9]. The order of events that transpire during this transient association is debated, although electron transfer, 2MgATP hydrolysis (to adenosine diphosphate, 2MgADP), the release of two inorganic phosphate (P i ) equivalents, and Fe protein dissociation occur.…”

“…Perhaps the two most-relevant redox couples of the P cluster (P N/1+ and P 1+/2+ ) both have E o 's of approximately −0.31 V vs. SHE. Further, the E o ' of the as-isolated FeMo-co (M N ) and its one-electron oxidation production (M OX ) is approximately −0.04 V vs. SHE, while a second lower-potential redox couple (M RED ) is also thought to be relevant to nitrogenase's catalytic cycle, although its E o´h as not been accurately determined [9].…”