Suppressing hydrogen peroxide generation to achieve oxygen-insensitivity of a [NiFe] hydrogenase in redox active films

Abstract

Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as hydrogenases for their use in fuel cells. However, the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day. Here, we employ operando confocal microscopy to elucidate the deactivation processes. The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst. We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] hydrogenase to over one week, approaching the experimental anaerobic half-life. Altogether, our data support the theory that redox films make the hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress.

The Solvent-Exposed Fe–S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni–Fe Carbon Monoxide Dehydrogenase

“…For certain metalloproteins, like the hydrogenases, oxidative inhibition has been well studied. 16,[23][24][25][26][27][28] For others, like the CODH family, investigations of the source of O 2 -sensitivity are just beginning.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…Alternatively, iodide was added in the whole system to induce dismutation of H 2 O 2 . An improvement of the half-life of the HASE in the presence of O 2 was demonstrated [295]. In the case of O 2 -tolerant HASEs, their specific sensitivity/tolerance to O 2 has been shown to lead to an even greater sensitivity to ROS.…”

“…Actually, low potential viologen-based redox polymers developed to protect HASEs against high potential inactivation also serve as shields against O 2 [224,237,288]. However, the viologen-based matrix itself may generate ROS in the course of the HASE protection mechanism, inducing its progressive decomposition [295]. To tackle this issue, a top polymer layer containing both GOx and catalase was used to ensure a complete HASE protection [143].…”

Reactivation of sulfide-protected [FeFe] hydrogenase in a redox-active hydrogel

“…5,8 Controlling the film thickness 9 enables optimization of the biocatalyst loading and life time, 10 reaching time scales of weeks under turnover and constant exposure to oxygen. 11 [FeFe] hydrogenases are considered to be the most active and reversible among the hydrogenase classes and, therefore, particularly relevant for biotechnological applications. 12 However, the high efficiency of [FeFe] hydrogenases comes with extreme oxygen sensitivity.…”

“…Iodide ions added to the electrolyte catalyze the decomposition of reactive oxygen species (generated from the viologen catalyzed O 2 reduction) to water preventing damage to the matrix, as reported previously. 11 The efficiency of the protection/reactivation procedure was demonstrated via cyclic voltammetry experiments. Redox hydrogel films were prepared by drop-casting a mixture of DdHydAB and a 2,2 0 -viologen modified polyvinylalcohol backbone 16 (P1) (Scheme S1, ESI †) on a glassy carbon electrode.…”