molybdenum/tungsten centre, Mo/W
6+ =S, is regenerated. Under non-steady-state
catalytic conditions (as the ones created in EPR experiments) the molybdenum/
tungsten one-electron oxidation should be favoured (Mo/W
4+
!Mo/W
5+ ), leading to
the formation of the EPR detectable Mo
5+
–SH species.
CO 2 reduction is suggested to follow the reverse reaction mechanism (Fig. 13,
green arrows). First, CO 2 binds to the reduced active site, not directly to the
molybdenum/tungsten ion, but at the same site as formate (and azide), with the
conserved arginine anchoring its oxygen atom(s) through hydrogen bond(s) and
orienting its carbon atom towards the protonated sulfido ligand. In an approximated way, based on the inhibition and Michaelis–Menten constants for the
D. desulfuricans FDH, the “binding strength” is suggested to follow the order
CO 2 (K m % 15 lM [137]) > azide (K i % 30 lM [136]) > formate (K m % 60 lM
[137]). Then, the reaction proceeds through straightforward hydride transfer from
the protonated sulfido group of the reduced molybdenum/tungsten centre,
Mo/W
4+
–SH, to the CO 2 carbon, whose LUMO have predominant C–p orbital
character, prone to nucleophile attack and reduction. This yields a formate moiety
and Mo/W
6+ =S. The subsequent re-reduction of Mo/W
6+ to Mo/W
4+ (via
intramolecular electron transfer from the enzyme physiological partner, through its
redox centre(s)) and formate release closes the catalytic cycle. The now reduced
Mo/W
4+ favours the sulfido group protonation and the initial reduced
molybdenum/tungsten centre, Mo/W
4+
–SH, is regenerated.
The FDH-catalysed reaction is reversible and the equilibrium between formate
oxidation versus CO 2 reduction is determined by the availability of formate versus
CO 2 and the ability to maintain the active site oxidised (Mo/W
6+ ) versus reduced
(Mo/W
4+ ), which, in its turn, determines the protonation state of the metal sulfido
group in a concerted and straightforward way.
Overall, the chemical strategy herein suggested is exactly the same as the one
proposed for the metal-independent FDHs: both bind formate in a close proximity
to an oxidised, electrophilic, hydride acceptor, which in metal-independent
enzymes is a NAD
+ molecule and in metal-dependent enzymes is the M
6+ =S
group; both bind CO 2 in a close proximity of a reduced, nucleophilic, hydride
donor, a NADH molecule or the M
4+
–SH group.
As expected, this mechanistic proposal faces some criticism and the most relevant one concerns the role of the active site selenocysteine/cysteine residue. In fact,
although the mechanism is suggested to operate in a hexa-coordinated metal centre
(Fig. 13), it can also take place in a penta-coordinated centre (Fig. 10), with an
unbound selenocysteine/cysteine—the sixth ligand does not seem to interfere with
the hydride transfer
12 . Even though there are experimental evidences (as discussed
above) and mechanistic arguments can be envisaged to support the necessity of
having a bound selenocysteine/cysteine (as discussed in [96, 98]), in the absence of
12 It should be noted that, xanthine oxidase, for example, that also uses a terminal sulfido group
as the hydride acceptor in the conversion of xanthine to urate, has a molybdenum
penta-coordinated active site, with no amino acid residues bound to it (the molybdenum ion is
coordinated by the cis-dithiolene (–S–C = C–S–) group of one pyranopterin cofactor molecule, the
terminal sulfido group plus two oxo groups (see previous Footnotes and references in the text).
Carbon Dioxide Utilisation—The Formate Route
55
6+ =S, is regenerated. Under non-steady-state
catalytic conditions (as the ones created in EPR experiments) the molybdenum/
tungsten one-electron oxidation should be favoured (Mo/W
4+
!Mo/W
5+ ), leading to
the formation of the EPR detectable Mo
5+
–SH species.
CO 2 reduction is suggested to follow the reverse reaction mechanism (Fig. 13,
green arrows). First, CO 2 binds to the reduced active site, not directly to the
molybdenum/tungsten ion, but at the same site as formate (and azide), with the
conserved arginine anchoring its oxygen atom(s) through hydrogen bond(s) and
orienting its carbon atom towards the protonated sulfido ligand. In an approximated way, based on the inhibition and Michaelis–Menten constants for the
D. desulfuricans FDH, the “binding strength” is suggested to follow the order
CO 2 (K m % 15 lM [137]) > azide (K i % 30 lM [136]) > formate (K m % 60 lM
[137]). Then, the reaction proceeds through straightforward hydride transfer from
the protonated sulfido group of the reduced molybdenum/tungsten centre,
Mo/W
4+
–SH, to the CO 2 carbon, whose LUMO have predominant C–p orbital
character, prone to nucleophile attack and reduction. This yields a formate moiety
and Mo/W
6+ =S. The subsequent re-reduction of Mo/W
6+ to Mo/W
4+ (via
intramolecular electron transfer from the enzyme physiological partner, through its
redox centre(s)) and formate release closes the catalytic cycle. The now reduced
Mo/W
4+ favours the sulfido group protonation and the initial reduced
molybdenum/tungsten centre, Mo/W
4+
–SH, is regenerated.
The FDH-catalysed reaction is reversible and the equilibrium between formate
oxidation versus CO 2 reduction is determined by the availability of formate versus
CO 2 and the ability to maintain the active site oxidised (Mo/W
6+ ) versus reduced
(Mo/W
4+ ), which, in its turn, determines the protonation state of the metal sulfido
group in a concerted and straightforward way.
Overall, the chemical strategy herein suggested is exactly the same as the one
proposed for the metal-independent FDHs: both bind formate in a close proximity
to an oxidised, electrophilic, hydride acceptor, which in metal-independent
enzymes is a NAD
+ molecule and in metal-dependent enzymes is the M
6+ =S
group; both bind CO 2 in a close proximity of a reduced, nucleophilic, hydride
donor, a NADH molecule or the M
4+
–SH group.
As expected, this mechanistic proposal faces some criticism and the most relevant one concerns the role of the active site selenocysteine/cysteine residue. In fact,
although the mechanism is suggested to operate in a hexa-coordinated metal centre
(Fig. 13), it can also take place in a penta-coordinated centre (Fig. 10), with an
unbound selenocysteine/cysteine—the sixth ligand does not seem to interfere with
the hydride transfer
12 . Even though there are experimental evidences (as discussed
above) and mechanistic arguments can be envisaged to support the necessity of
having a bound selenocysteine/cysteine (as discussed in [96, 98]), in the absence of
12 It should be noted that, xanthine oxidase, for example, that also uses a terminal sulfido group
as the hydride acceptor in the conversion of xanthine to urate, has a molybdenum
penta-coordinated active site, with no amino acid residues bound to it (the molybdenum ion is
coordinated by the cis-dithiolene (–S–C = C–S–) group of one pyranopterin cofactor molecule, the
terminal sulfido group plus two oxo groups (see previous Footnotes and references in the text).
Carbon Dioxide Utilisation—The Formate Route
55