crystallographic structures so far available, for FDH and FMFDH (Figs. 4, 5, 6 and
9, and references herein), show a stable hexa-coordination, with the cysteine or
selenocysteine always bound to the molybdenum/tungsten ion. This is also the case
of the recently solved structure of the formate-reduced D. vulgaris SeCys–W–FDH
[132] and also of the NADH-reduced R. capsulatus Cys–Mo–FDH, whose structure
was determined by cryo-electron microscopy [139].
XAS: Two recent XAS studies at the Mo K-edge suggested that, in R. capsulatus
Cys–Mo–FDH, the Mo
5+ state holds the cysteine residue bound to the metal, as the
oxidised Mo
6+ one, with a Mo-S(Cys) bond of 2.63 Å, while the Mo
4+ state of
formate-reduced enzyme has its cysteine displaced form the metal [169, 210].
However, contrary results were obtained with SeCys–Mo–FDHs from E. coli [213]
and D. desulfuricans [214], which showed a metal bound residue in all oxidised and
reduced states: the E. coli enzyme EXAFS data at both the Mo and Se K-edges were
interpreted as indicating the presence of one Mo–Se bond of 2.62 Å, plus one Se–S
bond of 2.19 Å (between the sulfido group and the selenocysteine selenium) [213].
EPR spectroscopy: Further experimental evidence for the stable
molybdenum/tungsten hexa-coordination came from EPR spectroscopy that clearly
showed that the selenocysteine/cysteine must remain bound to the Mo
5+ centre of
formate-reduced enzyme [208]. When the EPR spectrum is obtained from
77 Se-enriched enzyme, a very strong and anisotropic interaction with selenium is
observed (A 1,2,3 (
77 Se) = 13.2, 75, 240 MHz) [166]. This interaction and the
observation of the expected
95,97
Mo hyperfine coupling confirms that the selenium
atom of the selenocysteine is directly coordinated to the Mo
5+ and further suggests
that the unpaired electron is delocalised over the selenium (17–27%) and molybdenum atoms (73–83%) [166]. Also, the hydrogen atoms of the b-methylene carbon of
the selenocysteine residue are thought to be in the close proximity of the molybdenum atom, being responsible for an interaction with a not solvent-exchangeable
protons (A 1 = 35.1 MHz) [136]. Photolysis assays additionally confirmed that the
selenium/sulfur ligation is retained in the FDH Mo
5+ centre (the light beam did not
affect the strong selenium–molybdenum EPR interaction observed in
77
Se-enriched
FDH)
8 [166]. The Mo
5+ hexa-coordination (resulting from having the selenocysteine/
cysteine residue bound to the molybdenum ion) was also supported by theoretical
calculations on the signals-giving species of FDHs [215].
Inhibition assays: A different type of experimental evidence came from inhibition studies with iodoacetamide, an alkylating agent that reacts with “free” ionised
selenocysteine or cysteine residues (carboxamidomethylation). Native E. coli
SeCys–Mo–FDH H and its cysteine mutant [216] and native R. capsulatus Cys–
Mo–FDH [183] are not inhibited by iodoacetamide treatment. However, when the
preliminary iodoacetamide treatment (incubation) is carried out in the presence of
formate (not under turnover), both native and cysteine-containing mutant E. coli
8 These photolysis assays also demonstrate that the selenocysteine residue is not the formate Ca
hydrogen acceptor [166]: while the light beam did not affect the
77
Se interaction, it induced the
photolysis of the solvent-exchangeable formate-derived proton, showing that the selenocysteine
residue does not bind the strongly coupled proton mentioned above.
Carbon Dioxide Utilisation—The Formate Route
51
9, and references herein), show a stable hexa-coordination, with the cysteine or
selenocysteine always bound to the molybdenum/tungsten ion. This is also the case
of the recently solved structure of the formate-reduced D. vulgaris SeCys–W–FDH
[132] and also of the NADH-reduced R. capsulatus Cys–Mo–FDH, whose structure
was determined by cryo-electron microscopy [139].
XAS: Two recent XAS studies at the Mo K-edge suggested that, in R. capsulatus
Cys–Mo–FDH, the Mo
5+ state holds the cysteine residue bound to the metal, as the
oxidised Mo
6+ one, with a Mo-S(Cys) bond of 2.63 Å, while the Mo
4+ state of
formate-reduced enzyme has its cysteine displaced form the metal [169, 210].
However, contrary results were obtained with SeCys–Mo–FDHs from E. coli [213]
and D. desulfuricans [214], which showed a metal bound residue in all oxidised and
reduced states: the E. coli enzyme EXAFS data at both the Mo and Se K-edges were
interpreted as indicating the presence of one Mo–Se bond of 2.62 Å, plus one Se–S
bond of 2.19 Å (between the sulfido group and the selenocysteine selenium) [213].
EPR spectroscopy: Further experimental evidence for the stable
molybdenum/tungsten hexa-coordination came from EPR spectroscopy that clearly
showed that the selenocysteine/cysteine must remain bound to the Mo
5+ centre of
formate-reduced enzyme [208]. When the EPR spectrum is obtained from
77 Se-enriched enzyme, a very strong and anisotropic interaction with selenium is
observed (A 1,2,3 (
77 Se) = 13.2, 75, 240 MHz) [166]. This interaction and the
observation of the expected
95,97
Mo hyperfine coupling confirms that the selenium
atom of the selenocysteine is directly coordinated to the Mo
5+ and further suggests
that the unpaired electron is delocalised over the selenium (17–27%) and molybdenum atoms (73–83%) [166]. Also, the hydrogen atoms of the b-methylene carbon of
the selenocysteine residue are thought to be in the close proximity of the molybdenum atom, being responsible for an interaction with a not solvent-exchangeable
protons (A 1 = 35.1 MHz) [136]. Photolysis assays additionally confirmed that the
selenium/sulfur ligation is retained in the FDH Mo
5+ centre (the light beam did not
affect the strong selenium–molybdenum EPR interaction observed in
77
Se-enriched
FDH)
8 [166]. The Mo
5+ hexa-coordination (resulting from having the selenocysteine/
cysteine residue bound to the molybdenum ion) was also supported by theoretical
calculations on the signals-giving species of FDHs [215].
Inhibition assays: A different type of experimental evidence came from inhibition studies with iodoacetamide, an alkylating agent that reacts with “free” ionised
selenocysteine or cysteine residues (carboxamidomethylation). Native E. coli
SeCys–Mo–FDH H and its cysteine mutant [216] and native R. capsulatus Cys–
Mo–FDH [183] are not inhibited by iodoacetamide treatment. However, when the
preliminary iodoacetamide treatment (incubation) is carried out in the presence of
formate (not under turnover), both native and cysteine-containing mutant E. coli
8 These photolysis assays also demonstrate that the selenocysteine residue is not the formate Ca
hydrogen acceptor [166]: while the light beam did not affect the
77
Se interaction, it induced the
photolysis of the solvent-exchangeable formate-derived proton, showing that the selenocysteine
residue does not bind the strongly coupled proton mentioned above.
Carbon Dioxide Utilisation—The Formate Route
51