Mechanistic insight into the blocking of CO diffusion in [NiFe]-hydrogenase mutants through multiscale simulation

Abstract

[NiFe]-hydrogenases are fascinating biological catalysts with potential application in biofuel cells. However, a severe problem in practical application is the strong sensitivity of hydrogenase to gaseous inhibitor molecules such as CO and O 2 . Recently, a number of successful protein engineering studies have been reported that aimed at lowering the access of diatomic inhibitors to the active site pocket, but the molecular mechanism conferring increased resistance remained unclear. Here we use a multiscale simulation approach combining molecular dynamics with a master equation formalism to explain the steady drop in CO diffusion rate observed for the mutants V74M L122A, V74M L122M, and V74M of Desulfovibrio fructosovorans [NiFe]-hydrogenase. We find that diffusion in these variants is controlled by two gates, one between residues 74 and 476 and the other between residues 74 and 122. The existence of two control points in different locations explains why the reduction in the experimental diffusion rate does not simply correlate with the width of the main gas channel. We also find that in the more effective mutation (V74M) CO molecules are still able to reach the active site through transitions that are gated by the microsecond dihedral motions of the side chain of R476 and the thermal fluctuations of the width of the gas channel defined by M74 and L122. Reflecting on the molecular information gained from simulation, we discuss future mutation experiments that could further lower the diffusion rates of small ligands inhibiting [NiFe]-hydrogenase.gas diffusion | mean first passage times T here is currently a strong research effort in developing biology-inspired approaches for the production of renewable energy sources. Examples include the use of natural or artificial photosystems for light harvesting and exciton generation (1, 2), hydrogenases for hydrogen production (3, 4) and oxidation in biofuel cells (5, 6), and carbon-monoxide dehydrogenase for CO 2 reduction and synthesis of precursors of biofuels (7, 8). Yet, a general problem associated with the use of biocatalysts is their sensitivity to changing environmental conditions. A good example is the inhibition of hydrogenases by diatomic molecules. Naturally evolved under anaerobic conditions, the enzyme becomes inhibited by molecular oxygen that is present in the atmosphere and only few species can resist oxidative damage. Thus, an important aspect of biotechnological approaches is the ability to design and engineer enzyme variants that are more robust and durable than their natural counterparts.In the field of hydrogenase research, three strategies have been proposed to reduce the effect of inhibition or damage (5): (i) restricting the access of gas molecules to the active site, (ii) lowering the binding affinity of the gas molecules to the metal centers, and (iii) facilitating the removal of undesired oxidation products. Recently, evidence was found that oxygen-tolerance in the membrane-bound [NiFe]-hydrogenase from the Knallgas bacterium Ralstonia eutropha...

Mechanism of O2 diffusion and reduction in FeFe hydrogenases

“…The rate k i of conversion from A to I in eq 1 can be calculated from the rate constants in the following scheme: 24,25 $$\mathrm{boldF}\mathrm{bolde}\mathrm{boldF}\mathrm{bolde}\left(\mathrm{A}\right)\underset{k_{-1}}{\overset{k_{+1}\left[{\mathrm{normalO}}_2\right]}{\rightleftharpoons }}\mathrm{boldF}\mathrm{bolde}\mathrm{boldF}\mathrm{bolde}\dots {\mathbf{O}}_2\left(\mathrm{G}\right)\underset{k_{-2}}{\overset{k_{+2}}{\rightleftharpoons }}\mathrm{boldF}\mathrm{bolde}\mathrm{boldF}\mathrm{bolde}-{\mathbf{O}}_2\left(\mathrm{I}\right)$$Above, k +1 is the second-order rate constant for the diffusion of O 2 from the solvent to the active site and k − 1 is the first-order rate constant for the diffusion in the opposite direction. In the resulting state G (for “Geminate”), O 2 is in the active site pocket but it is not yet chemically bound to the active site.…”

Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

“…This is a kinetic two-step model consisting of an initial diffusion step of the gas molecule, followed by a metal-gas molecule chemical reaction step (Wang et al., 2011, Wang and Blumberger, 2012). Here, a classical force-field (FF) was employed, and only the diffusion and binding of O 2 gas molecules to the RNR iron center were modeled:$\mathrm{RNR}\left(\mathrm{Fe}\right)+{\mathrm{normalO}}_2\underset{{\text{k}}_{-1}}{\overset{{\text{k}}_{+1}}{\rightleftarrows }}\mathrm{RNR}\left(\mathrm{Fe}\right)\cdots {\mathrm{normalO}}_2.$…”

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

“…The nuanced role of the protein matrix in influencing catalysis is not restricted to oxygen delivery. In hydrogenases, gas channels have been reported to selectively filter molecules such as oxygen and carbon monoxide to prevent inactivation at the active site (12,17,35). Recent investigation of heme nitric oxide/oxygen binding domains has suggested that tunnels not only direct gases to the heme iron but also modulate reversible gas binding (16).…”