No abstract

Binding and activation of CO by nitrogenase is a topic of interest because CO is isoelectronic to N , the physiological substrate of this enzyme. The catalytic relevance of one- and multi-CO-bound states (the lo-CO and hi-CO states) of V-nitrogenase to C-C coupling and N reduction was examined. Enzymatic and spectroscopic studies demonstrate that the multiple CO moieties in the hi-CO state cannot be coupled as they are, suggesting that C-C coupling requires further activation and/or reduction of the bound CO entity. Moreover, these studies reveal an interesting correlation between decreased activity of N reduction and increased population of the lo-CO state, pointing to the catalytic relevance of the belt Fe atoms that are bridged by the single CO moiety in the lo-CO state. Together, these results provide a useful framework for gaining insights into the nitrogenase-catalyzed reaction via further exploration of the utility of the lo-CO conformation of V-nitrogenase.

Nitrogenase uses a reductase component called Fe protein to deliver electrons to its catalytic partner for substrate reduction. The essential role of Fe protein in catalysis makes it an ideal target for regulating the electron flux and enzymatic activity of nitrogenase without perturbing the cofactor site. This work reports that hybrids between the Fe protein homologs of Methanosarcina acetivorans and the catalytic components of Azotobacter vinelandii can trap substrate CO through reduced electron fluxes. In addition, homology modeling/in silico docking is used to define markers for binding energy and specificity between the component proteins that correlate with the experimentally determined activities. This homologue-based approach could be further developed to allow identification or design of hybrids between homologous nitrogenase components for mechanistic investigations of nitrogenase through capture of substrates/ intermediates or for transgenic expression of nitrogenase through synthetic biology.

The Fe protein of nitrogenase reduces two C1 substrates, CO 2 and CO, under ambient conditions when its [Fe 4 S 4 ] cluster adopts the all-ferrous [Fe 4 S 4 ] 0 state. Here, we show disparate reactivities of the nifH - and vnf -encoded Fe proteins from Methanosarcina acetivorans (designated Ma NifH and Ma VnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO 2 and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that Ma VnfH adopts the all-ferrous state at a more positive reduction potential than Ma NifH, which could account for the weaker reactivity of the Ma VnfH toward C1 substrates than Ma NifH. More importantly, Ma VnfH already displays the g = 16.4 parallel-mode EPR signal that is characteristic of the all-ferrous [Fe 4 S 4 ] 0 cluster at a reduction potential of −0.44 V, and the signal reaches 50% maximum intensity at a reduction potential of −0.59 V, suggesting the possibility of this Fe protein to access the all-ferrous [Fe 4 S 4 ] 0 state under physiological conditions. These results bear significant relevance to the long-lasting debate of whether the Fe protein can utilize the [Fe 4 S 4 ] 0/2+ redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes.

Nitrogenase iron (Fe) proteins reduce CO2 to CO and/or hydrocarbons under ambient conditions. Here, we report a 2.4-Å crystal structure of the Fe protein from Methanosarcina acetivorans (MaNifH), which is generated in the presence of a reductant, dithionite, and an alternative CO2 source, bicarbonate. Structural analysis of this methanogen Fe protein species suggests that CO2 is possibly captured in an unactivated, linear conformation near the [Fe4S4] cluster of MaNifH by a conserved arginine (Arg) pair in a concerted and, possibly, asymmetric manner. Density functional theory calculations and mutational analyses provide further support for the capture of CO2 on MaNifH while suggesting a possible role of Arg in the initial coordination of CO2 via hydrogen bonding and electrostatic interactions. These results provide a useful framework for further mechanistic investigations of CO2 activation by a surface-exposed [Fe4S4] cluster, which may facilitate future development of FeS catalysts for ambient conversion of CO2 into valuable chemical commodities. IMPORTANCE This work reports the crystal structure of a previously uncharacterized Fe protein from a methanogenic organism, which provides important insights into the structural properties of the less-characterized, yet highly interesting archaeal nitrogenase enzymes. Moreover, the structure-derived implications for CO2 capture by a surface-exposed [Fe4S4] cluster point to the possibility of developing novel strategies for CO2 sequestration while providing the initial insights into the unique mechanism of FeS-based CO2 activation.