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

## TL;DR

Researchers developed a fast method to find better cobalt-based proteins for producing hydrogen, a clean energy source.

## Contribution

A scalable workflow combining in vivo Co-PPIX incorporation and a colorimetric assay for screening hydrogen-producing proteins.

## Key findings

- The variant Co-Mut25 showed double the activity of the wild type in the screening assay.
- Co-Mut25 produced over 70% more hydrogen than the wild type when measured by gas chromatography.

## Abstract

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 b562 (cyt b562) 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.