molybdenum/tungsten centre or it would depend on the dissociation of the
cysteine/selenocysteine residue from the metal, in order to create a vacant position
(penta-coordinated centre) for substrate binding. While the hypothesis of the
hepta-coordinated metal centre was (so far) never perused, the dissociation of the
cysteine/selenocysteine is, as discussed above, controversial.
The two recent XAS studies mentioned above in point (vi) suggested that, in the
presence of formate, the cysteine ligation of (active) R. capsulatus Cys–Mo–FDH is
replaced by a long Mo–O bond of 2.15 Å, which was interpreted as arising from the
Mo–OCO(H) complex [169, 210]. The strong and competitive inhibition of E. coli
SeCys–Mo–FDH H-catalysed formate oxidation by azide, cyanate, thiocyanate,
nitrite and nitrate (1–00 lM range) was also evoked to support that formate, as well
as those inhibitors, bind directly to the molybdenum ion [219]. Yet, competitive
inhibition can arise if the inhibitor binds in the active site, but not directly to the
metal
10 [220]; this seems to be the case at least of azide, a well documented
inhibitor of both metal-independent [104, 171, 177] and metal-dependent [136,
166] FDHs (as suggested by EPR [136, 215]) and nitrite (as suggested by crystallography
11 ). Regarding nitrate, (once more) several other FDH enzymes are not
inhibited or the inhibition constants are 2–3 orders of magnitude higher than
K m (formate) [132, 135, 136, 166], or it is though as a substrate (even though a very
poor one; see Footnote 9 [182]). Moreover, the same study [219] showed that the
inhibition of the E. coli SeCys-Mo-FDH H-catalysed CO 2 reduction by those
anions is very weak (in the range of 1–50 mM) and not competitive in nature,
results that are contradictory to the hypothesis that the reduced active site (the one
that reacts with CO 2 ) becomes penta-coordinated, with an unbound selenocysteine
residue, and with an available position to bind inhibitors and CO 2 .
Therefore, except from the abovementioned XAS data, there are no other direct
experimental evidences of the direct formate or CO 2 binding to the FDH
molybdenum/tungsten ion; namely, there are no crystallographic structures showing
the formate molecule in the active site and there are no EPR signals showing the
presence of formate in the first coordination sphere of molybdenum/tungsten [136,
166, 184].
10 The active site conserved arginine residue, below suggested to be key to “anchor” formate and
CO 2 during turnover, could also be involved in the binding of these inhibitory anions through
electrostatic interactions—to have a strong and competitive inhibition of the formate oxidation, it
is not necessary that those anions bind directly to the molybdenum/tungsten ion it self.
The observed very weak inhibition of the CO 2 reduction by those anions (mentioned below in
this point (vii)) could be explained by subtle conformational changes involving the conserved
histidine residue upon reduction of the active site. Such conformational changes were described for
D. vulgaris FDH [132] and could explain why those inhibitory anions would not be stabilised at
the arginine spot within the reduced active site. Therefore, both the formate oxidation (strong
inhibition) and the CO 2 reduction (weak inhibition) could be inhibited without evoking the direct
binding of the inhibitory anions to the metal or the dissociation of the selenocysteine residue.
11 Boyington et al. [116] described the structure of E. coli SeCys-Mo-FDH treated with the
inhibitor nitrite as showing the selenocysteine bound to the molybdenum ion and the nitrite
molecule with one of its oxygen atoms at 2.58Å from the molybdenum.
Carbon Dioxide Utilisation—The Formate Route
53
cysteine/selenocysteine residue from the metal, in order to create a vacant position
(penta-coordinated centre) for substrate binding. While the hypothesis of the
hepta-coordinated metal centre was (so far) never perused, the dissociation of the
cysteine/selenocysteine is, as discussed above, controversial.
The two recent XAS studies mentioned above in point (vi) suggested that, in the
presence of formate, the cysteine ligation of (active) R. capsulatus Cys–Mo–FDH is
replaced by a long Mo–O bond of 2.15 Å, which was interpreted as arising from the
Mo–OCO(H) complex [169, 210]. The strong and competitive inhibition of E. coli
SeCys–Mo–FDH H-catalysed formate oxidation by azide, cyanate, thiocyanate,
nitrite and nitrate (1–00 lM range) was also evoked to support that formate, as well
as those inhibitors, bind directly to the molybdenum ion [219]. Yet, competitive
inhibition can arise if the inhibitor binds in the active site, but not directly to the
metal
10 [220]; this seems to be the case at least of azide, a well documented
inhibitor of both metal-independent [104, 171, 177] and metal-dependent [136,
166] FDHs (as suggested by EPR [136, 215]) and nitrite (as suggested by crystallography
11 ). Regarding nitrate, (once more) several other FDH enzymes are not
inhibited or the inhibition constants are 2–3 orders of magnitude higher than
K m (formate) [132, 135, 136, 166], or it is though as a substrate (even though a very
poor one; see Footnote 9 [182]). Moreover, the same study [219] showed that the
inhibition of the E. coli SeCys-Mo-FDH H-catalysed CO 2 reduction by those
anions is very weak (in the range of 1–50 mM) and not competitive in nature,
results that are contradictory to the hypothesis that the reduced active site (the one
that reacts with CO 2 ) becomes penta-coordinated, with an unbound selenocysteine
residue, and with an available position to bind inhibitors and CO 2 .
Therefore, except from the abovementioned XAS data, there are no other direct
experimental evidences of the direct formate or CO 2 binding to the FDH
molybdenum/tungsten ion; namely, there are no crystallographic structures showing
the formate molecule in the active site and there are no EPR signals showing the
presence of formate in the first coordination sphere of molybdenum/tungsten [136,
166, 184].
10 The active site conserved arginine residue, below suggested to be key to “anchor” formate and
CO 2 during turnover, could also be involved in the binding of these inhibitory anions through
electrostatic interactions—to have a strong and competitive inhibition of the formate oxidation, it
is not necessary that those anions bind directly to the molybdenum/tungsten ion it self.
The observed very weak inhibition of the CO 2 reduction by those anions (mentioned below in
this point (vii)) could be explained by subtle conformational changes involving the conserved
histidine residue upon reduction of the active site. Such conformational changes were described for
D. vulgaris FDH [132] and could explain why those inhibitory anions would not be stabilised at
the arginine spot within the reduced active site. Therefore, both the formate oxidation (strong
inhibition) and the CO 2 reduction (weak inhibition) could be inhibited without evoking the direct
binding of the inhibitory anions to the metal or the dissociation of the selenocysteine residue.
11 Boyington et al. [116] described the structure of E. coli SeCys-Mo-FDH treated with the
inhibitor nitrite as showing the selenocysteine bound to the molybdenum ion and the nitrite
molecule with one of its oxygen atoms at 2.58Å from the molybdenum.
Carbon Dioxide Utilisation—The Formate Route
53