Introduction of Methionines in the Gas Channel Makes [NiFe] Hydrogenase Aero-Tolerant

Abstract

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

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).…”

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

“…3A). The amount of nickel species corresponds to 20 -80% that of total nickel, depending on the enzyme preparation, the remaining nickel being EPR-silent (37). The ratio of NiB over NiA also strongly varies between preparations (37).…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…This was based on the observation that two highly conserved residues (valine 74 and leucine 122) at the end of the channel in O 2 -sensitive Hases are replaced by more bulky residues (isoleucine and phenylalanine) in regulatory Hases that control Hase gene expression and are resistant to O 2 . [123] Depending on the mutation, mutants of valine 74 were shown to either decrease the rate of O 2 diffusion to the active site, [124,125] or even to tend to acquire the phenotypes of O 2 -sensitive Hases, most probably because of faster electron-transfer rate between the active site and the closest FeS cluster (proximal cluster). [126,127] Reduction of the channel size is however not sufficient to provide O 2 tolerance.…”