Understanding and Harnessing Hydrogenases, Biological Dihydrogen Catalysts - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 116

atom at the H 2 -activating Fe guanylylpyridinol (FeGP) catalytic site. There are thus
two moles of Fe per mole of 76 kDa homodimer. Several [Fe] hydrogenase crystal
structures have now been resolved, revealing details about both the geometry of the
active site and inhibitor binding [ 69 , 70 ]. Based on these studies it is predicted that
upon H 2 binding the Fe is octahedrally coordinated by a Cys sulfur, two cis -CO
ligands, the pyridinol nitrogen and acyl carbon of the FeGP cofactor, with H 2 trans
to the acyl group.
The high O 2 sensitivity of this class of hydrogenase complicates experimental
work, and EPR cannot be used to characterize this class of enzymes because the Fe
remains in a low spin diamagnetic Fe 2+ oxidation state. However a recent study
comprising IR, NMR, and mass spectrometry analysis of the isolated cofactor
probed the origin of several parts of the active site and showed that the CO ligands
are derived from CO 2 [ 71 ]. Complementary deletion strain studies have also shown
that in addition to the hmd gene which encodes the [Fe] hydrogenases seven hcg
genes are essential for synthesis of a functional enzyme [ 72 ]. However, relative to
the biosynthesis of other hydrogenases, the mechanism for construction of the
FeGP cofactor is poorly understood and the biochemical activity of each accessory
protein is yet to be determined.

5 Insights into Hydrogenase Mechanism

from Small Molecule Mimics

Although within the context of Biology the presence of CO as an active site ligand
is a unique requirement of hydrogenases, within a chemical context CO is an
extremely common component of organometallic compounds. We rationalize that
CO is required to coordinate to Fe in the hydrogenase active site because its
ˀ

-acceptor bonding properties aid the stabilization of Fe in low oxidation states,
thus making Fe behave more like Pt [ 73 ]. Based on such a mechanistic understand-
ing of the reactivity properties of hydrogenase active sites it has been possible to
synthesize mimic compounds which both resemble hydrogenases and are active H 2
catalysts [ 45 , 74 ].
An advantage of studying synthetic small compound enzyme analogues rather
than complete enzymes is the greater number of analytical methods which can be
readily applied to understanding the structure and mechanism. For example, proton
nuclear magnetic resonance and neutron-scattering analysis techniques both
directly image hydride moieties but these experimental techniques cannot be
readily used to study large enzymes. In silico hydrogenase molecules can also be
created and computational mechanistic studies have been very valuable in
interpreting enzymatic spectral data and isolating reaction intermediates which
would be too short-lived to trap in vitro [ 45 , 75 ].