High-Throughput Screening of Co-Protoporphyrin IX-Binding Proteins for Enhanced Hydrogen Production

Artificial metalloenzymes incorporating cobalt protoporphyrin IX (Co-PPIX) are promising for sustainable hydrogen production; however, slow protein preparation and a lack of suitable detection methods limit the systematic optimization of their catalytic performance. Here, we report a streamlined workflow that combines the direct in vivo incorporation of Co-PPIX into cytochrome <i>b</i>₅₆₂ (cyt <i>b</i>₅₆₂) variants with a colorimetric assay for hydrogen evolution, scalable to hundreds of mutants. We screened 103 members of a mutant library and selected the variant Co-Mut25, which displayed activity double than wild type on the screen, and produced over 70% more hydrogen than WT as assessed by gas chromatography. This approach enables the rapid and scalable identification of high-performing cobalt-protein catalysts and expands the toolkit for artificial hydrogenase development.