Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases.

The kinetics of hydrogen oxidation and evolution by [FeFe]-hydrogenases have been investigated by electrochemical impedance spectroscopy-resolving factors that determine the exceptional activity of these enzymes, and introducing an unusual and powerful way of analyzing their catalytic electron transport properties. Attached to an electrode, hydrogenases display reversible electrocatalytic behavior close to the 2H/H potential, making them paradigms for efficiency: the electrocatalytic "exchange" rate (measured around zero driving force) is therefore an unusual parameter with theoretical and practical significance. Experiments were carried out on two [FeFe]-hydrogenases, HydA1 from the green alga , which contains only the active-site "H cluster," and I from the fermentative anaerobe , which contains four low-potential FeS clusters that serve as an electron relay in addition to the H cluster. Data analysis yields catalytic exchange rates (at the formal 2H/H potential, at 0 °C) of 157 electrons (78 molecules H) per second for I and 25 electrons (12 molecules H) per second for HydA1. The experiments show how the potential dependence of catalytic electron flow comprises frequency-dependent and frequency-independent terms that reflect the proficiencies of the catalytic site and the electron transfer pathway in each enzyme. The results highlight the "wire-like" behavior of the Fe-S electron relay in I and a low reorganization energy for electron transfer on/off the H cluster.