[NiFe] hydrogenases catalyze the reversible cleavage of molecular hydrogen into protons and electrons. Here, we have studied the impact of temperature and illumination on an oxygen-tolerant and thermostable [NiFe] hydrogenase by IR and EPR spectroscopy. Equilibrium mixtures of two catalytic [NiFe] states, Nia-C and Nia-SR’’, were found to drastically change with temperature, indicating a thermal exchange of electrons between the [NiFe] active site and iron-sulfur clusters of the enzyme. In addition, IR and EPR experiments performed under illumination revealed an unusual photochemical response of the enzyme. Nia-SR’’, a fully reduced hydride intermediate of the catalytic cycle, was found to be reversibly photoconverted into another catalytic state, Nia-L. In contrast to the well-known photolysis of the more oxidized hydride intermediate Nia-C, photoconversion of Nia-SR’’ into Nia-L is an active-site redox reaction that involves light-driven electron transfer towards the enzyme's iron-sulfur clusters. Omitting the ground-state intermediate Nia-C, this direct interconversion of these two states represents a potential photochemical shortcut of the catalytic cycle that integrates multiple redox sites of the enzyme. In total, our findings reveal the non-local redistribution of electrons via thermal and photochemical reaction channels and the potential of accelerating or controlling [NiFe] hydrogenases by light.
