Abstract
ABSTRACTThe Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.
Highlights
IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid
Using this A. vinelandii strain, a V-cluster-containing native form of the VFe protein was produced in vivo when V was supplemented in the growth medium (Fig. 1A), where deletion of the Mo transporter prevented incorporation of trace Mo into the cofactor [12,13,14], whereas an M-cluster-containing hybrid form of the VFe protein was produced in vivo when Mo was added in excess to the growth medium (Fig. 1A), where the uptake of Mo was accomplished by other transporter systems, such as those involving siderophores [15, 16]
VnfDGKM by the M-cluster, further highlighting the characteristics of the unique properties of the M-cluster in the reaction of carbon monoxide (CO) reduction. These results suggest a combined effect of protein environment and cofactor properties on the reactivity of nitrogenase toward CO: the protein scaffold has a significant impact on the overall activity of CO reduction (Fig. 4C, VnfDGKM versus NifDKM), whereas the cofactor species fine-tunes the product profile of CO reduction while exerting a moderate impact on the overall activity (Fig. 4C, VnfDGKV versus VnfDGKM)
Summary
IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. ® mbio.asm.org 1 strates at the cofactor site of the catalytic component upon accumulation of sufficient electrons [2, 3] Using this two-component mechanism, the nitrogenase is capable of reducing nitrogen (N2) to ammonia (NH3), as well as carbon monoxide (CO) to hydrocarbons (e.g., propane [C3H8] and butane [C4H10]) [4, 5] at ambient conditions. These two reactions parallel the industrial Haber-Bosch and Fischer-Tropsch processes, respectively, which are used for large-scale production of ammonia and carbon fuels. The observation of highly differential CO-reducing activities of two homologous nitrogenases has prompted us to define key features of these systems that contribute to this discrepancy in activity; in particular, the question of whether the protein environment or the cofactor species determines the reactivity of nitrogenase toward CO needs to be addressed, as knowledge in this regard represents the first step toward understanding the CO-reducing activity of nitrogenase for the potential applications of this reactivity in the future
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