Theoretically, it can be argued that, since the FDH-catalysed reaction does not
involve the transfer of an oxygen atom (as explained above in point (ii)), there is no
need to form the otherwise expected Mo/W
6+
–OCO(H) or Mo/W
4+
–OCO complexes (follow Mo
4+
–OR and Mo
4+
–OQ in Fig. 12). It can also be argued in the
opposite way: if formate/CO 2 binds directly to the molybdenum/tungsten ion, why
there is no oxygen atom transfer to form hydrogencarbonate (Eq. 7)? Overall, in the
absence of more definitive experimental evidences, we must continue to ask: Does
the direct formate/CO 2 binding to the metal occur? Is it necessary or desirable to
interconvert formate and CO 2 ? Is the penta-coordinated metal centre with unbound
cysteine/selenocysteine catalytically relevant?
(c) Currently accepted mechanistic hypotheses
In accordance with the well-established points (i) to (v) highlighted above, the
FDH-catalysed formate oxidation and CO 2 reduction are presently recognised to
occur through hydride transfer (Eq. 9), with the oxidised and reduced active site
sulfido group, Mo/W
6+ =S and Mo/W
4+
–SH, acting as the direct hydride acceptor
and donor, respectively. Yet, points (vi) and (vii) still raise questions to some
authors regarding the coordination of the active site and substrates binding during
FDH catalysis.
As originally proposed by Niks et al. [184] for formate oxidation and shortly
after also for CO 2 reduction [137], we suggest that FDH catalysis proceeds as
follows (the reaction mechanism is suggested to be identical in Mo–FDH and W–
FDH, as well as in FMFDH):
Formate oxidation (Fig. 13, blue arrows) is initiated with the formate binding to
the oxidised active site, but not directly to the molybdenum/tungsten atom. Following the example provided by the metal-independent FDH, where the
formate-binding site harbours arginine and asparagine residues [102–109], it is
suggested that the conserved arginine residue is essential to drive the formate Ca
hydrogen towards the sulfido ligand, by establishing hydrogen bond(s) with its
oxygen atom(s). Also, azide (N 3
− , isoelectronic with CO 2 ) is suggested to bind
(tightly) to the same site and not directly to the molybdenum/tungsten ion (as had
been previously suggested for the D. desulfuricans FDH inhibition by azide [136,
215]). The binding of azide and formate to the same site, and not to the
molybdenum/tungsten atom itself, explains why azide is a powerful inhibitor of both
metal-independent (K i = 40 nM for Candida boidinii) [104, 171, 177] and metaldependent FDHs [136, 166]. A similar reasoning applies to the inhibitor nitrite
(isoelectronic with formate). Formate oxidation, then, proceeds by a straightforward
hydride transfer from formate to the sulfido group of the oxidised molybdenum/
tungsten centre, Mo/W
6+ =S, leading to the formation of Mo/W
4+
–SH and CO 2 . The
re-oxidation of Mo/W
4+ to Mo/W
6+ (via intramolecular electron transfer to the
enzyme other(s) redox centre(s) and, eventually, to the physiological partner) and the
release of CO 2 close the catalytic cycle. The now oxidised Mo/W
6+ favours the sulfido
group deprotonation (dictated by the ligand pK a [205–207]), and the initial oxidised
54
L. B. Maia et al.
involve the transfer of an oxygen atom (as explained above in point (ii)), there is no
need to form the otherwise expected Mo/W
6+
–OCO(H) or Mo/W
4+
–OCO complexes (follow Mo
4+
–OR and Mo
4+
–OQ in Fig. 12). It can also be argued in the
opposite way: if formate/CO 2 binds directly to the molybdenum/tungsten ion, why
there is no oxygen atom transfer to form hydrogencarbonate (Eq. 7)? Overall, in the
absence of more definitive experimental evidences, we must continue to ask: Does
the direct formate/CO 2 binding to the metal occur? Is it necessary or desirable to
interconvert formate and CO 2 ? Is the penta-coordinated metal centre with unbound
cysteine/selenocysteine catalytically relevant?
(c) Currently accepted mechanistic hypotheses
In accordance with the well-established points (i) to (v) highlighted above, the
FDH-catalysed formate oxidation and CO 2 reduction are presently recognised to
occur through hydride transfer (Eq. 9), with the oxidised and reduced active site
sulfido group, Mo/W
6+ =S and Mo/W
4+
–SH, acting as the direct hydride acceptor
and donor, respectively. Yet, points (vi) and (vii) still raise questions to some
authors regarding the coordination of the active site and substrates binding during
FDH catalysis.
As originally proposed by Niks et al. [184] for formate oxidation and shortly
after also for CO 2 reduction [137], we suggest that FDH catalysis proceeds as
follows (the reaction mechanism is suggested to be identical in Mo–FDH and W–
FDH, as well as in FMFDH):
Formate oxidation (Fig. 13, blue arrows) is initiated with the formate binding to
the oxidised active site, but not directly to the molybdenum/tungsten atom. Following the example provided by the metal-independent FDH, where the
formate-binding site harbours arginine and asparagine residues [102–109], it is
suggested that the conserved arginine residue is essential to drive the formate Ca
hydrogen towards the sulfido ligand, by establishing hydrogen bond(s) with its
oxygen atom(s). Also, azide (N 3
− , isoelectronic with CO 2 ) is suggested to bind
(tightly) to the same site and not directly to the molybdenum/tungsten ion (as had
been previously suggested for the D. desulfuricans FDH inhibition by azide [136,
215]). The binding of azide and formate to the same site, and not to the
molybdenum/tungsten atom itself, explains why azide is a powerful inhibitor of both
metal-independent (K i = 40 nM for Candida boidinii) [104, 171, 177] and metaldependent FDHs [136, 166]. A similar reasoning applies to the inhibitor nitrite
(isoelectronic with formate). Formate oxidation, then, proceeds by a straightforward
hydride transfer from formate to the sulfido group of the oxidised molybdenum/
tungsten centre, Mo/W
6+ =S, leading to the formation of Mo/W
4+
–SH and CO 2 . The
re-oxidation of Mo/W
4+ to Mo/W
6+ (via intramolecular electron transfer to the
enzyme other(s) redox centre(s) and, eventually, to the physiological partner) and the
release of CO 2 close the catalytic cycle. The now oxidised Mo/W
6+ favours the sulfido
group deprotonation (dictated by the ligand pK a [205–207]), and the initial oxidised
54
L. B. Maia et al.