A Conserved Glutamate Orchestrates Transitions Between Catalytic Intermediates in [NiFe]-Hydrogenase

Abstract

[NiFe]-hydrogenases catalyze the reversible cleavage of molecular hydrogen with exceptional efficiency under mild conditions and therefore serve as powerful blueprints for the development of sustainable, bioinspired H2-evolving catalysts. While the structure of the NiFe(CN)2CO active site has been extensively characterized, how outer-sphere residues regulate catalytic dynamics and proton-coupled electron transfer remains poorly understood. Here, we examine the functional role of a strictly conserved glutamate residue in the second coordination sphere of the regulatory [NiFe]-hydrogenase from Cupriavidus necator. Substitution of this glutamate with glutamine results in a dramatic loss (>99%) of catalytic activity. However, comprehensive IR, EPR, and resonance Raman spectroscopic analyses reveal that the residue is not required for the formation or stabilization of the key catalytic intermediates along the Nia-S → Nia-SR → Nia-C → Nia-L1 sequence. Notably, low-temperature IR spectroscopy shows that the transition from Nia-L1 to Nia-L2 is selectively disrupted in the absence of the conserved glutamate. These results identify the Nia-L2 state as a bona fide catalytic intermediate and demonstrate that the glutamate residue initiates critical outer-sphere rearrangements required to advance the catalytic cycle and enable productive proton transfer. Together, these findings elucidate how the protein matrix actively controls active-site reactivity in [NiFe]-hydrogenases and highlight the importance of second-sphere interactions in tuning catalytic efficiency. This work provides mechanistic principles that are directly relevant to the rational design of synthetic and biomimetic hydrogen- evolving catalysts for sustainable energy conversion.