Carbon Monoxide. Toxic Gas and Fuel for Anaerobes and Aerobes: Carbon Monoxide Dehydrogenases - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 45

The active site is buried within the L subunit. It is accessible from the surface
through a hydrophobic channel with a diameter of 6 , which ends at the [CuSMo]

unit of the active site. The enedithiolate unit of the pyranopterin cofactor binds Mo,
whereas Cu is coordinated by the cysteine sulfur of the active site loop. Cu and Mo
are bridged by a

Å

ʼ 2 -sulfido ligand, keeping Cu and Mo in a distance of 3.74 Å in the

Mo(VI) state, which increases to 3.93 in the Mo(IV) state [ 50 ].

The ligands of Mo have a distorted square pyramidal arrangement. The equatorial
coordination sphere of the Mo ion consists of the enedithiolate moiety of the pyran
ring, the bridging sulfido ligand, and one oxo/hydroxyl (or hydroxo) ligand.
An additional oxo ligand is found in the apical position. A glutamate residue in
trans to the apical oxo ligand with a Glu-Mo distance of 3.14

Å

and a glutamine
residue in hydrogen-bonding distance to the apical oxo ligand create the second
coordination sphere of Mo (Figure 3 ). All elements of the first and second coordina-
tion sphere are conserved in the Mo hydroxylases.
The [2Fe2S] clusters and FAD are bound in the S and M subunit, respectively. The
[2Fe2S] cluster proximal to the active site Mo is bound in a four-helix bundle domain,
while the distal [2Fe2S] cluster is coordinated within a domain closely resembling the
fold of a plant-type ferredoxin [ 49 ]. Both [2Fe2S] clusters are shielded from the
solvent. The FAD molecule is bound between the N-terminal and middle domain of
the M subunit. Access to the redox-active N5 position of the isoalloxazine ring of
FAD is restricted by a tyrosine in the O. carboxydovorans and a tryptophan residue
in the H. pseudoflava Cu,Mo-CODH [ 49 , 52 ]. The cofactors are arranged in two
independent electron transfer chains in the dimer with short cofactor-cofactor
distances within each monomer allowing a rapid electron transfer [ 54 ].

Å

2.1.2 Spectroscopic Investigations

Molybdenum cycles through three oxidation states (Mo(VI), Mo(V) and Mo(IV))
during catalysis of which only Mo(V) is paramagnetic and exhibits an EPR signal.
The Mo(V) signal of Cu,Mo-CODH shows strong hyperfine coupling with Cu
(I ¼ 3/2) [ 55 ], which is consistent with a delocalization of the electron spin within
the SOMO of Mo(V) along the entire Mo(V)-S-Cu(I) moiety [ 56 ]. This strong
coupling is reproduced in a Mo-S-Cu model complex with a geometry similar to
the active site of Cu,Mo-CODH [ 57 ]. The Mo(V) signal splits upon addition of
[ 13 C]CO indicating that either [ 13 C]CO or product [ 13 C]CO 2 is a part of the signal-
giving species [ 55 ]. An extended analysis of the [ 13 C]CO- 63,65 Cu coupling using
EPR and ENDOR spectroscopy provided additional evidence for the presence of a
copper-carbonyl intermediate in CO oxidation [ 56 ]. That the Mo(V) signal changes
in the presence of substrate has also been observed for H 2 . When Cu,Mo-CODH
reacts with H 2 a new EPR signal with larger g anisotropy and hyperfine coupling to
the 63,65 Cu in the active site arises [ 58 ].
Confirmatory and complementary insights have also been obtained by X-ray
absorption spectroscopy, which confirmed the presence of Cu and Mo in the active
site of Cu,Mo-CODH [ 59 ]. While the crystallographic study indicates the presence