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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]