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of a hydroxyl group in the equatorial plane, X-ray absorption spectroscopy studies
are consistent with the presence of an oxo group [ 59 ], which is also favored by
computational studies on the mechanism of Cu,Mo-CODH [ 56 , 60 , 61 ].
2.1.3 Enzymatic Activity
Further details of the catalytic cycle were revealed by determining the activity of
Cu,Mo-CODH with different substrates. At its pH optimum of 7.2 Cu,Mo-CODH
oxidizes CO with a k cat of 93.3 s 1 and a K m for CO of 10.7 ʼ Mat25 C[ 55 ]. Quasi
single-turnover kinetics (up to three turnovers) indicate that the likely rate limiting
step of the overall reaction is the reductive half-reaction and that CO binds in a
rapid initial step to the enzyme. Furthermore, Cu,Mo-CODH acts as an efficient
uptake hydrogenase [ 62 ] oxidizing H 2 with a limiting rate constant of 5.3 s 1 and a
K d for H 2 of 525
M[ 58 ].
Cu,Mo-CODH transfers electrons generated by CO oxidation to different
physiological and artificial electron acceptors [ 39 , 48 ]. In O. carboxydovorans
cells the enzyme is attached to the inner aspect of the cytoplasmic membrane [ 63 ]
and the interaction is specific for cytoplasmic membranes of CO-grown cells [ 64 ].
Electrons generated by CO-oxidation at the Cu,Mo-site are transferred efficiently
from the FAD to membrane-bound quinones, agreeing with a direct electron
transfer into the quinone pool under physiological conditions [ 53 ].
Cu,Mo-CODHs are inactivated by several small molecule inhibitors. Cyanide
is isoelectronic and isosteric with CO and inactivates the oxidized enzyme with a
half-life of about 30 mins. Cyanolysis releases concomitantly up to 1 mol of thiocy-
anate (SCN ) and 1 mol of Cu per active site of Cu,Mo-CODH [ 50 ]. The crystal
structure and spectroscopic investigations are in agreement with the formation of a Mo
tri-oxo species upon cyanolysis, depleting the active site of Cu and the bridging
sulfido-ligand [ 50 , 59 ]. Isocyanides and CO show a similar
ʼ
˃
π
-acceptor
ligand character and are isoelectronic with a non-bonding pair of electrons in the
p-orbital of the terminal carbon. Isocyanides act as inhibitors, which bind to the active
site of Cu,Mo-CODH by inserting into the Cu-S bond. Inhibition is concurrent with
oxidation of the inhibitor, which forms a thiocarbamate derivative [ 50 ]. The inactive
Mo tri-oxo species is also formed when isolated Cu,Mo-CODH reacts with CO under
oxic conditions, indicating that the rapid transfer of the electrons generated by CO
oxidation to the quinone pool [ 53 ] is important to render the enzyme stable under
physiological conditions when turnover occurs in the presence of dioxygen.
Inactive Mo tri-oxo Cu,Mo-CODH can be reconstituted by addition of sulfide
and copper under anoxic, reducing conditions, resulting in about 50 % functional
enzyme species [ 65 ]. A reconstitution protocol similar to that of Resch, Dobbek,
and Meyer [ 65 ] was used to substitute copper for silver, generating a Ag,Mo-CODH
[ 66 ]. Most surprisingly, activity of the enzyme was regained and resulted in approx.
30 % functional enzyme after reconstitution. The silver-substituted enzyme has a
limiting rate constant of 8.1 s 1 in CO oxidation [ 66 ], but unlike the Cu-containing
enzyme it is unable to oxidize H 2 [ 58 ].
-donor and
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