Structural Analysis of a Nitrogenase Iron Protein from Methanosarcina acetivorans: Implications for CO2 Capture by a Surface-Exposed [Fe4S4] Cluster.

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

Genetic and Chemical Alterations Affecting the Activity of Nitrogenase

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

Evaluation of instrumented shoes for ambulatory assessment of ground reaction forces

Currently, force plates or pressure sensitive insoles are the standard tools to measure ground reaction forces and centre of pressure data during human gait. Force plates, however, impose constraints on foot placement, and the available pressure sensitive insoles measure only one component of force. In this study, shoes instrumented with two force transducers measuring forces and moments in three dimensions were evaluated. Technical performance was assessed by comparing force measurement and centre of pressure reconstructions of the instrumented shoes against a force plate. The effect of the instrumented shoes on gait was investigated using an optical tracking system and a force plate. Instrumented shoes were compared against normal shoes and weighted shoes. The ground reaction force measured with force plate and instrumented shoes differed by 2.2 ± 0.1% in magnitude and by 3.4 ± 1.3° in direction. The horizontal components differed by 9.9 ± 3.8% in magnitude and 26.9 ± 10.0° in direction. Centre of pressure location differed by 13.7 ± 2.4 mm between measurement systems. A MANOVA repeated measures analysis on data of seven subjects, revealed significant differences in gait pattern between shoe types (p ≤ 0.05). A subsequent univariate analysis showed significant differences only in maximum ground reaction force but these could not be attributed to specific shoe types by pair-wise comparison. This study indicates that shoes instrumented with force transducers can be a valuable alternative to current measurement systems if accurate sensing of position and orientation of the force transducers is improved. They are applicable in ambulatory settings and suitable for inverse dynamics analysis

Structural Analysis of a Nitrogenase Iron Protein from Methanosarcina acetivorans: Implications for CO2 Capture by a Surface-Exposed [Fe4S4] Cluster

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.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