In-vivo turnover frequency of the cyanobacterial NiFe-hydrogenase during photohydrogen production outperforms in-vitro systems.

Cyanobacteria provide all components for sunlight driven biohydrogen production. Their bidirectional NiFe-hydrogenase is resistant against low levels of oxygen with a preference for hydrogen evolution. However, until now it was unclear if its catalytic efficiency can keep pace with the photosynthetic electron transfer rate. We identified NikKLMQO (sll0381-sll0385) as a nickel transporter, which is required for hydrogen production. ICP-MS measurements were used to quantify hydrogenase molecules per cell. We found 400 to 2000 hydrogenase molecules per cell depending on the conditions. In-vivo turnover frequencies of the enzyme ranged from 62 H/s in the wild type to 120 H/s in a mutant during photohydrogen production. These frequencies are above maximum in-vivo photosynthetic electron transfer rates of 47 e/s (equivalent to 24 H/s). They are also above those of existing in-vitro systems working with unlimited electron supply and show that in-vivo photohydrogen production is limited by electron delivery to the enzyme.