molybdenum/tungsten ion [117, 169, 180–183]—an hypothesis discussed below
in point (vi). Photolysis assays with
77 Se-enriched FDH, described below in point
(vi) and Footnote 8, further confirmed that the selenocysteine residue cannot be
the hydrogen atom acceptor [166].
Further evidence that the sulfido group becomes protonated upon reduction was
also provided by a recent X-ray absorption spectroscopy (XAS) study with the R.
capsulatus Mo-FDH [210].
(v) The terminal sulfido group is essential to both formate oxidation and CO 2
reduction. It is well established, by numerous spectroscopic and kinetic studies,
that cyanide reacts with the active site sulfido group of different molybdoenzymes, such as xanthine oxidase, and abstracts it in the form of thiocyanate,
yielding a desulfo enzyme form that harbours an oxo group in the place of the
native sulfido group [110–112, 193–195, 198, 199, 202]. The sulfido by oxo
replacement renders xanthine oxidase and other enzymes inactive, because its
active site is no longer able to accept a hydride (see Footnote 6). The same and
complete cyanide inhibition is observed in several FDH, such as the ones from
Methanobacterium formicicum [211], Alcaligenes eutrophus [212], E. coli
[167], R. capsulatus (where the sulfido was observed to be replaced by an oxo
group) [210], or D. desulfuricans (where thiocyanate formation accounted to
0.87 per molybdenum atom) [137]. Together with the experimental evidences
that support the involvement of the sulfido group as a hydrogen atom acceptor
during FDH catalysis (described above), these inhibitory results demonstrate
that the sulfido group acts as a hydride acceptor/donor in FDH catalysis.
(b) Two interrelated points are not yet consensual
(vi) Does the active site cysteine or selenocysteine residue dissociate from the
metal during catalysis?
If the configuration of the oxidised active site is consensually accepted, the reduced
form still finds a few contradictory experimental evidences (Fig. 10).
X-ray crystallography: In a reinterpretation of the crystallographic data of the
reduced E. coli SeCys–Mo–FDH H originally obtained by Boyington et al. in
1997 [116], Raaijmakers and Romão in 2006 [117] suggested that the polypeptide
loop containing the selenocysteine was not properly traced in the original work and
that the selenocysteine residue is not bound to the metal, but, instead, is found
dissociated from the molybdenum ion and shifted away (12 Å) (Fig. 4). Therefore,
those authors suggested that, while in the oxidised state the selenocysteine residue
is coordinated to the metal, the enzyme reduction triggers the residue dissociation,
resulting in a square pyramidal penta-coordinated centre, where the molybdenum
ion is coordinated by the cis-dithiolene (–S–C = C–S–) group of two pyranopterin
cofactor molecules (in the equatorial positions) plus the terminal sulfido group (in
the axial position) (Fig. 10). Regardless of this reinterpretation, all other
50
L. B. Maia et al.
in point (vi). Photolysis assays with
77 Se-enriched FDH, described below in point
(vi) and Footnote 8, further confirmed that the selenocysteine residue cannot be
the hydrogen atom acceptor [166].
Further evidence that the sulfido group becomes protonated upon reduction was
also provided by a recent X-ray absorption spectroscopy (XAS) study with the R.
capsulatus Mo-FDH [210].
(v) The terminal sulfido group is essential to both formate oxidation and CO 2
reduction. It is well established, by numerous spectroscopic and kinetic studies,
that cyanide reacts with the active site sulfido group of different molybdoenzymes, such as xanthine oxidase, and abstracts it in the form of thiocyanate,
yielding a desulfo enzyme form that harbours an oxo group in the place of the
native sulfido group [110–112, 193–195, 198, 199, 202]. The sulfido by oxo
replacement renders xanthine oxidase and other enzymes inactive, because its
active site is no longer able to accept a hydride (see Footnote 6). The same and
complete cyanide inhibition is observed in several FDH, such as the ones from
Methanobacterium formicicum [211], Alcaligenes eutrophus [212], E. coli
[167], R. capsulatus (where the sulfido was observed to be replaced by an oxo
group) [210], or D. desulfuricans (where thiocyanate formation accounted to
0.87 per molybdenum atom) [137]. Together with the experimental evidences
that support the involvement of the sulfido group as a hydrogen atom acceptor
during FDH catalysis (described above), these inhibitory results demonstrate
that the sulfido group acts as a hydride acceptor/donor in FDH catalysis.
(b) Two interrelated points are not yet consensual
(vi) Does the active site cysteine or selenocysteine residue dissociate from the
metal during catalysis?
If the configuration of the oxidised active site is consensually accepted, the reduced
form still finds a few contradictory experimental evidences (Fig. 10).
X-ray crystallography: In a reinterpretation of the crystallographic data of the
reduced E. coli SeCys–Mo–FDH H originally obtained by Boyington et al. in
1997 [116], Raaijmakers and Romão in 2006 [117] suggested that the polypeptide
loop containing the selenocysteine was not properly traced in the original work and
that the selenocysteine residue is not bound to the metal, but, instead, is found
dissociated from the molybdenum ion and shifted away (12 Å) (Fig. 4). Therefore,
those authors suggested that, while in the oxidised state the selenocysteine residue
is coordinated to the metal, the enzyme reduction triggers the residue dissociation,
resulting in a square pyramidal penta-coordinated centre, where the molybdenum
ion is coordinated by the cis-dithiolene (–S–C = C–S–) group of two pyranopterin
cofactor molecules (in the equatorial positions) plus the terminal sulfido group (in
the axial position) (Fig. 10). Regardless of this reinterpretation, all other
50
L. B. Maia et al.