Hydride state accumulation in native [FeFe]-hydrogenase with the physiological reductant H(2) supports its catalytic relevance

Small molecules in solution may interfere with mechanistic investigations, as they can affect the stability of catalytic states and produce off-cycle states that can be mistaken for catalytically relevant species. Here we show that the hydride state (H(hyd)), a proposed central intermediate in the catalytic cycle of [FeFe]-hydrogenase, can be formed in wild-type [FeFe]-hydrogenases treated with H(2) in absence of other, non-biological, reductants. Moreover, we reveal a new state with unclear role in catalysis induced by common low pH buffers.