The unusual formaldehyde-induced activation of [NiFe]-hydrogenase: Implications from protein film electrochemistry and infrared spectroscopy.

Here we investigate how formaldehyde (HCHO), a known strong inhibitor of [FeFe]‑hydrogenases and a mild inhibitor of [NiFe]‑hydrogenases, may exert more complex effects on this group of metalloenzymes, which reversibly catalyze the 2H+/H₂ reaction. We investigated the [NiFe]‑hydrogenase Hyd-2 from E. coli using protein film electrochemistry, a technique that enables the measurement of enzyme activity when the enzyme is adequately adsorbed on the electrode. The effect of HCHO on the electrocatalytic performance of Hyd-2 is highly dependent on the buffer pH and the direction of catalysis. During H₂ production, HCHO consistently acts as an inhibitor of Hyd-2. However, this effect is reversed in acidic pH values, where HCHO can mildly enhance the electrocatalytic H₂ oxidation by Hyd-2. FTIR investigations did not detect any new redox intermediate resulting from the inhibition or activation. Therefore, we propose that HCHO - a natural electrophile that can readily react with nucleophiles and proton acceptors - may facilitate the transfer protons during the rapid transformation of different redox species participating in the catalytic cycle of [NiFe]‑hydrogenases.