[FeFe]-hydrogenases are highly efficient enzymes in the reversible catalysis of molecular hydrogen production and oxidation. Their active site, the H-cluster, consists of a [4Fe–4S]H subcluster linked to a binuclear [2Fe]H organometallic unit. Many [FeFe]-hydrogenases, such as the one from Desulfovibrio desulfuricans (DdHydAB), possess accessory Fe–S clusters (F and F′) that mediate electron transfer. This study employs hybrid quantum mechanics/molecular mechanics (QM/MM) methods to characterize the electronic structure and thermodynamic landscape associated with redox and protonation events in the complete Fe–S cluster network of DdHydAB. Our calculations indicate that the F′ cluster plays a key role in the initial reduction of the oxidized resting state, acting as the preferential site for the accumulation of the first electron. Analysis of protonated states, upon reduction events, reveals a strong correlation between protonation and electron transfer (PCET), with protonation at the H-cluster inducing electron transfer from the F′ cluster to the H-cluster. Calculations indicate that the formation of a terminal hydride is energetically favored over ADT protonation, and subsequent isomerization to a bridging hydride (μ-H) is further stabilizing, albeit potentially kinetically limiting. The study highlights how accessory clusters influence the electronic distribution and redox properties of the H-cluster, underscoring the importance of considering the entire Fe–S cluster system for a complete understanding of the catalytic mechanism of [FeFe]-hydrogenases.

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