40
2 Selective Production of Methanol …
A metal-binding site housing one copper ion has been found in the hydrophilic
N-terminal region of the polypeptide PmoB [82–85] and was first modeled as a dinuclear copper site [82]. However, a recent study using quantum mechanical refinement of X-ray crystallographic data suggested that the data were better fith by a
single copper center rather than two copper ions [92]. In fact, pMMO containing
only two mono-nuclear copper ions has been purified with retaining its activity [93].
In this book, this copper site is referred to as the PmoB–N copper site. An additional
copper-binding site is located in the transmembrane region of the PmoC polypeptide.
Copper ions bind to this site with lower affinity than to the PmoB–N copper site;
thus, the copper can be replaced by a zinc ion when pMMO is isolated from bacterial cells [85]. Thus, this site is referred to as a Cu/Zn site in this book. These two
copper-binding sites have been identified in a number of X-ray analyses of pMMO
protein crystals [82–85].
Several additional copper-binding sites other than the PmoB–N copper site and
Cu/Zn site have been identified via X-ray analysis of pMMO protein crystals; one is
located in the transmembrane region of the PmoA polypeptide [94–96], and another
is found in the C-terminal hydrophilic region of PmoB [97].
2.3.2 Catalytic Site
Among the metal-binding sites that have been identified in pMMO, the PmoB–N
site is considered the most probable catalytic site for the hydroxylation of methane,
because recombinant pMMO subdomain proteins containing only the PmoB–N copper site have shown the ability to oxidize methane to methanol [84, 90, 98]. At this
site, one or two copper ions are coordinated with a histidine residue and a nitrogen
atom from the terminal amino group of the peptide chain, as shown in Fig. 2.11.
2.3.3 Reaction Mechanism
The active oxygen species for methane hydroxylation should be produced via the
activation of molecular oxygen at the catalytic site of pMMO. Unfortunately, the
intermediate of pMMO bearing the active species has not been detected spectroscopically. Instead, quantum chemistry calculations based on the crystallographic
analysis of pMMO have been a useful tool for exploring the reaction mechanism,
although the calculation results depend on the experimental crystallographic data.
In a calculation based on a model with di-nuclear copper as site A, a mixed valence
(+2 and +3) oxohydroxo-bridged di-nuclear copper center (Cu
II Cu
III -(μ-O
− ) (μOH)) was proposed as the active species [99, 100]. The corresponding reaction
mechanism is shown schematically in Fig. 2.12.
In this reaction mechanism, the oxygen cross-linked to the metal ion is a radical
and extracts a hydrogen radical from the C–H bond of methane. OH and methyl
radicals cross-linked to metal ions are believed to be generated as a result, with
C–O bonds being formed via a “radical rebound mechanism” and “asynchronous
2 Selective Production of Methanol …
A metal-binding site housing one copper ion has been found in the hydrophilic
N-terminal region of the polypeptide PmoB [82–85] and was first modeled as a dinuclear copper site [82]. However, a recent study using quantum mechanical refinement of X-ray crystallographic data suggested that the data were better fith by a
single copper center rather than two copper ions [92]. In fact, pMMO containing
only two mono-nuclear copper ions has been purified with retaining its activity [93].
In this book, this copper site is referred to as the PmoB–N copper site. An additional
copper-binding site is located in the transmembrane region of the PmoC polypeptide.
Copper ions bind to this site with lower affinity than to the PmoB–N copper site;
thus, the copper can be replaced by a zinc ion when pMMO is isolated from bacterial cells [85]. Thus, this site is referred to as a Cu/Zn site in this book. These two
copper-binding sites have been identified in a number of X-ray analyses of pMMO
protein crystals [82–85].
Several additional copper-binding sites other than the PmoB–N copper site and
Cu/Zn site have been identified via X-ray analysis of pMMO protein crystals; one is
located in the transmembrane region of the PmoA polypeptide [94–96], and another
is found in the C-terminal hydrophilic region of PmoB [97].
2.3.2 Catalytic Site
Among the metal-binding sites that have been identified in pMMO, the PmoB–N
site is considered the most probable catalytic site for the hydroxylation of methane,
because recombinant pMMO subdomain proteins containing only the PmoB–N copper site have shown the ability to oxidize methane to methanol [84, 90, 98]. At this
site, one or two copper ions are coordinated with a histidine residue and a nitrogen
atom from the terminal amino group of the peptide chain, as shown in Fig. 2.11.
2.3.3 Reaction Mechanism
The active oxygen species for methane hydroxylation should be produced via the
activation of molecular oxygen at the catalytic site of pMMO. Unfortunately, the
intermediate of pMMO bearing the active species has not been detected spectroscopically. Instead, quantum chemistry calculations based on the crystallographic
analysis of pMMO have been a useful tool for exploring the reaction mechanism,
although the calculation results depend on the experimental crystallographic data.
In a calculation based on a model with di-nuclear copper as site A, a mixed valence
(+2 and +3) oxohydroxo-bridged di-nuclear copper center (Cu
II Cu
III -(μ-O
− ) (μOH)) was proposed as the active species [99, 100]. The corresponding reaction
mechanism is shown schematically in Fig. 2.12.
In this reaction mechanism, the oxygen cross-linked to the metal ion is a radical
and extracts a hydrogen radical from the C–H bond of methane. OH and methyl
radicals cross-linked to metal ions are believed to be generated as a result, with
C–O bonds being formed via a “radical rebound mechanism” and “asynchronous