UC Irvine

The bacterial enzyme nitrogenase regularly catalyses the reduction of dinitrogen (N2) to ammonia (NH3), thereby forming bioavailable nitrogen. The same enzyme can also reduce carbon monoxide (CO) to short chain hydrocarbons, turning pollutant gas into biofuel. For both reactions the catalytic NifDK relies on electron supply from the reductase NifH. The electron transfer is mediated by a Fe4S4 cluster on NifH, which also enables the reductase protein to perform catalysis of carbon dioxide (CO2) to CO and short chain hydrocarbons. NifDK utilizes a MoFe7S9C-(R)-homocitrate cluster, termed M-cluster, within its active site for reduction of the respective substrates. Reduction of CO2 by NifH from Methanosarcina acetivorans was investigated here. For this investigation, the protein was heterologously expressed in Escherichia coli and assessed for its ability to reduce CO2. In contrast to the NiH homologue from Azotobacter vinelandii, the most well-studied nitrogenase expressing bacterium, NifH from M. acetivorans reduced CO2 beyond CO to hydrocarbons. To further investigate this advanced catalysis by this NifH protein, specific point mutations of conserved arginine residues in close proximity to the active site on NifH were created. Assessment of the CO2-reduction capability of these mutant proteins highlighted the need for hydrogen-bonding and proton-donating residues in close proximity to the active site on NifH. In a different project, the role of (R)-homocitrate of the M-cluster of NifDK was investigated. By creating a genetic knockout of the homocitrate-synthase in A. vinelandii, (R)-homocitrate was made unavailable to the cell. This caused citrate to be integrated during the M-cluster synthesis as the organic ligand, which led to a lower N2-reduction but a higher CO-reduction rate by the NifDKCit protein. In vitro maturation of the M-cluster additionally allowed for the integration of isocitrate as the organic ligand of the M-cluster. Upon reconstitution of M-cluster-deficient NifDK with M-clusterIsocit, the enzyme also showed a lower N2-reduction but an even higher CO-reduction rate than NifDK reconstituted with regular M-cluster or M-clusterCit.