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Hydrogenases are metalloproteins capable of catalyzing the interconversion between molecular hydrogen and protons and electrons. The iron-sulfur clusters within the enzyme enable rapid relay of electrons which are either consumed or generated at the active site. Their unparalleled catalytic efficiency has attracted attention, especially for potential use in H 2 production and/or fuel cell technologies. However, there are limitations to using hydrogenases, especially due to their high O 2 sensitivity. The subclass, called [FeFe] hydrogenases, are particularly more vulnerable to O 2 but proficient in H 2 production. In this review, we provide an overview of mechanistic and protein engineering studies focused on understanding and enhancing O 2 tolerance of the enzyme. The emphasis is on ongoing studies that attempt to overcome O 2 sensitivity of the enzyme while it catalyzes H 2 production in an aerobic environment. We also discuss pioneering attempts to utilize the enzyme in biological H 2 production and other industrial processes, as well as our own perspective on future applications., (© 2019 Wiley Periodicals, Inc.)

Photosynthetic H 2 production has been a compelling but elusive objective. Here we describe how coordinated bioreactor, metabolic pathway, and protein engineering now suggest feasibility for the sustainable, solar-powered production of a storable fuel to complement our expanding photovoltaic and wind based capacities. The need to contain and harvest the gaseous products provides decisive solar bioreactor design advantages by limiting O 2 exposure to prolific, but O 2 -sensitive H 2 producing enzymes-[FeFe] hydrogenases. CO 2 supply and cell growth can also be limited so that most of the photosynthetic reduction capacity is directed toward H 2 production. Yet, natural [FeFe] hydrogenases are still too O 2 sensitive for technology implementation. We report the discovery of new variants and a new O 2 tolerance mechanism that significantly reduce the sensitivity to O 2 exposure without lowering H 2 production rates or losing electrons to O 2 reduction. Testing the improved hydrogenases with a biologically derived, light-dependent electron source provides evidence that this game changing technology has the potential for sustainable large-scale fuel production., (Copyright © 2018. Published by Elsevier Inc.)

Hydrogenases, ferredoxins, and ferredoxin-NADP + reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H 2 production technologies. A high-throughput H 2 production assay device (H 2 PAD) is presented that enables simultaneous evaluation of 96 individual H 2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H 2 PAD senses the chemo-optical response of Pd/WO 3 thin films to the H 2 produced. H 2 PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H 2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H 2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH-driven H 2 production rates. H 2 PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biological H 2 production is one of the alternative technologies for producing H 2 in a renewable and sustainable fashion. The technology often relies on the [FeFe] hydrogenase enzyme for catalyzing the H 2 production reaction from protons and electrons as it is highly efficient. The high O 2 sensitivity of the enzyme have been one of the key obstacles for implementing the technology, but recent findings showed the feasibility of improving O 2 tolerance via protein engineering. In this study, we investigated the changes in the O 2 tolerance and aerobic H 2 production activity of an [FeFe] hydrogenase by replacing methionines on the surface with leucines. None of the mutations were detrimental to protein folding, and a few were able to exert either positive or negative influence on the O 2 tolerance of the enzyme. We combined the mutation exhibiting the highest improvement in O 2 tolerance with the rest of the Met – Leu replacements; however, the results indicated non-synergistic impact on the tolerance. Combinations with the other types of mutations that have been reported previously also failed to further improve the O 2 tolerance of the hydrogenase. These results provide new insights on how mutagenesis affects the enzyme's functional properties, as well as the molecular mechanism(s) behind the tolerance. • A novel mutation that enhances the O 2 tolerance of the [FeFe] hydrogenase. • Analysis of the combinatorial mutagenesis' influence on the key functional properties. • Protein engineering work employing both in silico and experimental methods. [ABSTRACT FROM AUTHOR]

Hydrogenases are metalloproteins capable of catalyzing the interconversion between molecular hydrogen and protons and electrons. The iron–sulfur clusters within the enzyme enable rapid relay of electrons which are either consumed or generated at the active site. Their unparalleled catalytic efficiency has attracted attention, especially for potential use in H2 production and/or fuel cell technologies. However, there are limitations to using hydrogenases, especially due to their high O2 sensitivity. The subclass, called [FeFe] hydrogenases, are particularly more vulnerable to O2 but proficient in H2 production. In this review, we provide an overview of mechanistic and protein engineering studies focused on understanding and enhancing O2 tolerance of the enzyme. The emphasis is on ongoing studies that attempt to overcome O2 sensitivity of the enzyme while it catalyzes H2 production in an aerobic environment. We also discuss pioneering attempts to utilize the enzyme in biological H2 production and other industrial processes, as well as our own perspective on future applications. [ABSTRACT FROM AUTHOR]

Hydrogenases, ferredoxins, and ferredoxin-NADP+ reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H2 production technologies. A high-throughput H2 production assay device (H2PAD) is presented that enables simultaneous evaluation of 96 individual H2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H2PAD senses the chemo-optical response of Pd/WO3 thin films to the H2 produced. H2PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH-driven H2 production rates. H2PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems. [ABSTRACT FROM AUTHOR]