In vivo 2D-IR Spectroscopy of [NiFe] Hydrogenases: A Shielding Role of the Protein Matrix

Abstract

ABSTRACT: Hydrogenases are metalloenzymes that catalyze the cleavage and evolution of dihydrogen (H₂), a perfectly clean fuel. Thus, they represent ideal model catalysts for sustainable energy conversion approaches utilizing H₂. Due to the presence of biologically uncommon CO and CN⁻ ligands at their catalytic metal sites, infrared (IR) spectroscopy is a key technique in hydrogenase research that can even be used to study these enzymes within living cells. Here, we introduce two-dimensional (2D) IR spectroscopy as a new in vivo technique for exploring the impact of the conditions in the cytoplasm on the properties of hydrogenases. Utilizing the soluble NAD⁺-reducing [NiFe] hydrogenase from the H₂-oxidizing model bacterium Cupriavidus necator H16 as a suitable and biotechnologically relevant model enzyme, we demonstrate the feasibility of this approach. Our data indicates that even subtle structural details of the [NiFe] active site are unaffected by the unique gel-like properties of the highly dense cytoplasm, pointing towards a shielding role of the protein matrix that isolates this deeply buried metal center from environmental influences. In a more general sense, this study demonstrates that adequate strategies for scatter suppression can turn 2D-IR spectroscopy into a suitable technique for probing enzymes and other molecular targets in living cells and other complex biological environments