FDHs are inhibited [216]. Inhibition is also observed in the R. capsulatus FDH, but
only when the iodoacetamide treatment (incubation) is carried out in the presence of
nitrate; in this case, the cysteine carboxamidomethylation was confirmed by mass
spectroscopy [183]. On the other hand, native D. vulgaris SeCys–W–FDH is
inhibited by iodoacetamide, but mass spectroscopy clearly showed that the inhibition is not due to the carboxamidomethylation of the active site selenocysteine
residue (but of 9 other cysteine residues not present in the active site) [132]. In
addition, other FDHs are not at all affected by iodoacetamide [135, 136]. Hence, the
inhibition results available were not obtained under formate/CO 2 turnover conditions and the inconsistency of the results, once more, do not contribute to provide a
definitive answer.
Overall, the majority of experimental evidences points towards a
molybdenum/tungsten stable hexa-coordination, with the cysteine/selenocysteine
residue always bound to the metal. Regarding the results showing a metal
penta-coordination, with unbound cysteine/selenocysteine residue, it is possible that
the crystallisation/irradiation had induced some artefacts that are not relevant to the
enzyme activity; but it is also possible that the species crystallographically characterised, being catalytically relevant, bear no relation to the species observed by
XAS and EPR (with these being not catalytically relevant). Certainly,
high-resolution structures are needed to confirm the existence of the two alternating
conformations of the selenocysteine/cysteine-containing polypeptide loop and to
discuss the catalytic relevance of each conformation.
(vii) Do formate/CO 2 bind directly to the molybdenum/tungsten ion during
catalysis?
Inspired by the oxotransfer chemistry displayed by several molybdenum—and
tungsten-dependent enzymes (Fig. 12) [110–112, 162–165], and in particular by
periplasmatic nitrate reductases
9
, it was suggested that FDH catalysis necessarily
involves the formate/CO 2 direct binding to the molybdenum/tungsten ion [180, 218].
To begin the discussion of this point, it should be noted that the direct
formate/CO 2 binding would involve an unprecedented hepta-coordinated
9 The C. necator periplasmatic nitrate reductase catalyses the reduction of nitrate to nitrite
(ONO 2
− + 2e
−
+ 2H
+ ! NO 2
− + H 2 O), and it was described to share with FDHs the same
molybdenum coordination sphere, with the cis-dithiolene (–S–C = C–S–) group of two
pyranopterin cofactor molecules, one terminal sulfido group and one sulfur atom from a cysteine
residue [217]. The similarity in the active site metal centre led some authors to suggest similar
mechanistic strategies for nitrate reductases and FDHs—leading to the so-called sulfur-shift
mechanism [180, 218].
To give further support to the hypothesis of a similar mechanistic strategy, the nitrate reductase
activity of different FDHs was investigated. The R. capsulatus Cys-Mo-FDH was described to be
able to catalyse the nitrate reduction to nitrite, even though at an extremely low catalytic constant
(k cat (nitrate) = 0.21 min
−1 [183] that compares very poorly with k cat (formate) = 2124 min
−1 and
k cat (CO 2 ) = 89 min
−1 [182]), and the catalysis was suggested by XAS to involve the dissociation
of the cysteine residue from the molybdenum ion, with the subsequent nitrate binding [183].
However, other FDHs failed to reduce nitrate (such as the ones from D. desulfuricans [135, 136],
D. vulgaris [132]).
52
L. B. Maia et al.
only when the iodoacetamide treatment (incubation) is carried out in the presence of
nitrate; in this case, the cysteine carboxamidomethylation was confirmed by mass
spectroscopy [183]. On the other hand, native D. vulgaris SeCys–W–FDH is
inhibited by iodoacetamide, but mass spectroscopy clearly showed that the inhibition is not due to the carboxamidomethylation of the active site selenocysteine
residue (but of 9 other cysteine residues not present in the active site) [132]. In
addition, other FDHs are not at all affected by iodoacetamide [135, 136]. Hence, the
inhibition results available were not obtained under formate/CO 2 turnover conditions and the inconsistency of the results, once more, do not contribute to provide a
definitive answer.
Overall, the majority of experimental evidences points towards a
molybdenum/tungsten stable hexa-coordination, with the cysteine/selenocysteine
residue always bound to the metal. Regarding the results showing a metal
penta-coordination, with unbound cysteine/selenocysteine residue, it is possible that
the crystallisation/irradiation had induced some artefacts that are not relevant to the
enzyme activity; but it is also possible that the species crystallographically characterised, being catalytically relevant, bear no relation to the species observed by
XAS and EPR (with these being not catalytically relevant). Certainly,
high-resolution structures are needed to confirm the existence of the two alternating
conformations of the selenocysteine/cysteine-containing polypeptide loop and to
discuss the catalytic relevance of each conformation.
(vii) Do formate/CO 2 bind directly to the molybdenum/tungsten ion during
catalysis?
Inspired by the oxotransfer chemistry displayed by several molybdenum—and
tungsten-dependent enzymes (Fig. 12) [110–112, 162–165], and in particular by
periplasmatic nitrate reductases
9
, it was suggested that FDH catalysis necessarily
involves the formate/CO 2 direct binding to the molybdenum/tungsten ion [180, 218].
To begin the discussion of this point, it should be noted that the direct
formate/CO 2 binding would involve an unprecedented hepta-coordinated
9 The C. necator periplasmatic nitrate reductase catalyses the reduction of nitrate to nitrite
(ONO 2
− + 2e
−
+ 2H
+ ! NO 2
− + H 2 O), and it was described to share with FDHs the same
molybdenum coordination sphere, with the cis-dithiolene (–S–C = C–S–) group of two
pyranopterin cofactor molecules, one terminal sulfido group and one sulfur atom from a cysteine
residue [217]. The similarity in the active site metal centre led some authors to suggest similar
mechanistic strategies for nitrate reductases and FDHs—leading to the so-called sulfur-shift
mechanism [180, 218].
To give further support to the hypothesis of a similar mechanistic strategy, the nitrate reductase
activity of different FDHs was investigated. The R. capsulatus Cys-Mo-FDH was described to be
able to catalyse the nitrate reduction to nitrite, even though at an extremely low catalytic constant
(k cat (nitrate) = 0.21 min
−1 [183] that compares very poorly with k cat (formate) = 2124 min
−1 and
k cat (CO 2 ) = 89 min
−1 [182]), and the catalysis was suggested by XAS to involve the dissociation
of the cysteine residue from the molybdenum ion, with the subsequent nitrate binding [183].
However, other FDHs failed to reduce nitrate (such as the ones from D. desulfuricans [135, 136],
D. vulgaris [132]).
52
L. B. Maia et al.