# H‑cluster Intermediates and Catalytic Properties of Clostridium pasteurianum [FeFe]-Hydrogenase III

## TL;DR

This paper studies the unique H-cluster intermediates in Clostridium pasteurianum [FeFe]-hydrogenase III and how they differ from other similar enzymes.

## Contribution

The study identifies distinct redox and spectroscopic properties of the H-cluster in CpIII, revealing novel catalytic intermediates.

## Key findings

- CpIII's H-cluster resting state is fully oxidized, differing from typical [FeFe]-hydrogenases.
- Under H2, the Htrans-like state dominates over Hox, suggesting a unique role in catalysis.
- CpIII lacks spectral evidence for fully reduced states like HsredH+ and Hhyd.

## Abstract

[FeFe]-Hydrogenases are structurally diverse enzymes
that catalyze
reversible H2 activation at a catalytic cofactor or H-cluster.
The H-cluster is a [4Fe-4S] cubane linked by a cysteine thiolate to
a diiron subsite containing unique CO, CN-, and dithiomethylamine
ligands. The established H-cluster resting state of [4Fe-4S]2+-[FeII-FeI], or Hox, functions in
H2 binding and oxidation, or by proton-coupled reduction
initiates H2 evolution. In contrast, in Clostridium pasteurianum [FeFe]-hydrogenase III (CpIII)
the resting state of the H-cluster is fully oxidized, [4Fe-4S]2+-[FeII-FeII], or Hox+1.
To begin to understand if Hox+1 has a role in the mechanism
of CpIII, we determined the spectroscopic and redox properties of
CpIII H-cluster states under catalytic conditions. CpIII poised in
Hox+1 and either equilibrated under 1 atm of H2 or reduced with sodium dithionite, resulted in a mixture of reduced
states including Hox (E
m
8 = −407 mV), Htrans-like [4Fe-4S]+-[FeII-FeII] (E
m
8 = −418 mV), Hred [4Fe-4S]+-[FeII-FeI], and HredH+ [4Fe-4S]2+-[FeI-FeI] (E
m
8 = −455–480 mV). Under H2 the population of the Htrans-like state was >20-fold
higher than Hox, implicating a role in CpIII catalysis.
Unlike other enzymes, there was no spectral evidence of fully reduced
states, such as HsredH+ ([4Fe-4S]+-[FeI-FeI]) or Hhyd ([4Fe-4S]+-[FeII‑FeII]-H–). Thus, while
the H-cluster states of CpIII encompass most of the catalytic intermediates,
it is either unable to form HsredH+ and Hhyd, or these states are highly destabilized in CpIII. Thus, these results
demonstrate that catalytic intermediates of reduced CpIII differ from
the typical intermediates of other catalytic [FeFe]-hydrogenases and
may explain the catalytic preference for H2 production.