Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates

[FeFe] hydrogenases are extremely active H2-converting
enzymes. Their mechanism remains highly controversial, in particular,
the nature of the one-electron and two-electron reduced intermediates
called HredH+ and HsredH+. In one model, the HredH+ and HsredH+ states contain a semibridging CO, while in the other
model, the bridging CO is replaced by a bridging hydride. Using low-temperature
IR spectroscopy and nuclear resonance vibrational spectroscopy, together
with density functional theory calculations, we show that the bridging
CO is retained in the HsredH+ and HredH+ states in the [FeFe] hydrogenases from Chlamydomonas
reinhardtii and Desulfovibrio desulfuricans, respectively. Furthermore, there is no evidence for a bridging
hydride in either state. These results agree with a model of the catalytic
cycle in which the HredH+ and HsredH+ states are integral, catalytically competent components.
We conclude that proton-coupled electron transfer between the two
subclusters is crucial to catalysis and allows these enzymes to operate
in a highly efficient and reversible manner.