Hydride state accumulation in native [FeFe]-hydrogenase with the physiological reductant H2 supports its catalytic relevance

Small molecules in solution may 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 (Hhyd), a proposed central intermediate in the catalytic cycle of [FeFe] hydrogenase, can be formed in wild-type [FeFe]-hydrogenases treated with H2 in strict absence of other, non-biological, reductants. Moreover, we reveal a new state with unclear role in catalysis induced by common low pH buffers.