Reconsidering the Enzyme Kinetics of [FeFe]-Hydrogenases: Improved Turnover Rates and New Insights into pH and Potential Dependence with Eu(II)-Based Solution Assays

Metal-dependent redox enzymes are central for microbial processing of gases, as exemplified by hydrogenase, nitrogenase, and carbon monoxide dehydrogenase. Due to their remarkable efficiencies and high biotechnological relevance, such gas-processing enzymes are intensively studied. Nevertheless, many of their mechanistic details remain opaque. We herein report a new method for solution assays under reducing conditions based on europium(II) as a terminal reductant and show how it can be employed to gain new insight into hydrogenase kinetics. Compared with the commonly used reductant sodium dithionite, this work shows that Eu(II) can serve as a robust and relatively easy-to-handle alternative electron donor, also providing a larger potential window for catalytic studies. Further, this work clarifies previous discrepancies in the literature regarding the influence of pH on hydrogenase kinetics in these assays. Our study shows that sodium dithionite, most likely due to its decomposition into SO₂, alters hydrogenase kinetics in solution assays. Using [FeFe]-hydrogenase I from <i>Clostridium pasteurianum</i> (CpI) as a model system, Eu(II)-based solution assays demonstrated a pH optimum of 5-6 and rates greatly exceeding those observed with sodium dithionite assays. The higher turnover frequencies observed at low pH obtained with Eu(II) align more closely with the electrochemical data. Additionally, a strong driving force dependency was identified. A solution potential change of approximately 180 mV resulted in a 35-fold increase in the catalytic rate, yielding activities far surpassing those of earlier reports on CpI turnover frequencies. These findings provide new insight into the pH dependence and overall kinetic performance of [FeFe]-hydrogenases. More broadly, the report outlines alternative assay methods employing Eu(II) to better understand the enzyme kinetics of hydrogenases and related metalloenzymes.