36
2 Selective Production of Methanol …
The dissociation constant between MMOH and MMOR was measured by monitoring the quenching of the tryptophan fluorescence spectrum. Based on these measurements, two distinct dissociation constants (K d ) of approximately 10 nM and 8 μM
were estimated [66]. The larger K d value was reproduced in a fluorescence anisotropy
titration of the formation of the complex between MMOH and the ferredoxin domain
of MMOR in the presence of MMOB [67], while a smaller value (0.9 μM) was measured in the absence of MMOB [63]. The results imply that MMOR and MMOB
share the same binding site on the surface of MMOH.
2.2.7 Control of the Catalytic Cycle via the Interaction
of MMOB with MMOH
Electron transfer from MMOR, access of oxygen and methane molecules to the
di-nuclear iron site, and control of the dissociation of the product methanol from
MMOH are achieved by the interaction of the small protein MMOB with MMOH
and MMOR. The mechanisms by which MMOB controls the oxidation of methane
to methanol are described in this section.
MMOB has been reported to have the following effects in the catalytic cycle of
methane hydroxylation in MMOH:
(a) Increasing the turnover number of MMOH 150-fold and the rate of the initial
reaction with O 2 by a factor of 1000 [9, 12].
(b) Decreasing the redox potential of the di-nuclear iron center of MMOH
[15, 68, 69].
(c) Diminishing the rate of intermolecular electron transfer between MMOR and
MMOH [70].
(d) Altering the conversion rate of methane and C2–C8 n-alkanes and changing
product regioselectivity [22, 71, 72].
These effects of MMOB on the enzymatic properties of MMOH arise from specific
features of the interaction of MMOB with MMOH. Therefore, the structure and
function of MMOB have been investigated intensively in order to obtain a better
understanding of the mechanism of methane hydroxylation by sMMO.
The results of various experiments, including saturation-recovery EPR spectroscopy of spin-labeled MMOB [73], matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) peptide identification of chemical cross-linking
between MMOH and MMOB [74, 75], and crystallographic analysis of the MMOH–
MMOB complex [58, 76] have shown that MMOB interacts with MMOH at the
canyon-like cavity, as shown in Fig. 2.10.
In particular, crystallographic data derived from the MMOH–MMOB complex
provided detailed information regarding the conformational changes in the protein
structure of MMOH after the binding of MMOB. MMOB has an unstructured
N-terminal peptide chain (Met1–Ser35), a well-folded core region (Asp36–Leu129)
with seven β-strands and three α-helices, and a short C-terminal sequence
2 Selective Production of Methanol …
The dissociation constant between MMOH and MMOR was measured by monitoring the quenching of the tryptophan fluorescence spectrum. Based on these measurements, two distinct dissociation constants (K d ) of approximately 10 nM and 8 μM
were estimated [66]. The larger K d value was reproduced in a fluorescence anisotropy
titration of the formation of the complex between MMOH and the ferredoxin domain
of MMOR in the presence of MMOB [67], while a smaller value (0.9 μM) was measured in the absence of MMOB [63]. The results imply that MMOR and MMOB
share the same binding site on the surface of MMOH.
2.2.7 Control of the Catalytic Cycle via the Interaction
of MMOB with MMOH
Electron transfer from MMOR, access of oxygen and methane molecules to the
di-nuclear iron site, and control of the dissociation of the product methanol from
MMOH are achieved by the interaction of the small protein MMOB with MMOH
and MMOR. The mechanisms by which MMOB controls the oxidation of methane
to methanol are described in this section.
MMOB has been reported to have the following effects in the catalytic cycle of
methane hydroxylation in MMOH:
(a) Increasing the turnover number of MMOH 150-fold and the rate of the initial
reaction with O 2 by a factor of 1000 [9, 12].
(b) Decreasing the redox potential of the di-nuclear iron center of MMOH
[15, 68, 69].
(c) Diminishing the rate of intermolecular electron transfer between MMOR and
MMOH [70].
(d) Altering the conversion rate of methane and C2–C8 n-alkanes and changing
product regioselectivity [22, 71, 72].
These effects of MMOB on the enzymatic properties of MMOH arise from specific
features of the interaction of MMOB with MMOH. Therefore, the structure and
function of MMOB have been investigated intensively in order to obtain a better
understanding of the mechanism of methane hydroxylation by sMMO.
The results of various experiments, including saturation-recovery EPR spectroscopy of spin-labeled MMOB [73], matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) peptide identification of chemical cross-linking
between MMOH and MMOB [74, 75], and crystallographic analysis of the MMOH–
MMOB complex [58, 76] have shown that MMOB interacts with MMOH at the
canyon-like cavity, as shown in Fig. 2.10.
In particular, crystallographic data derived from the MMOH–MMOB complex
provided detailed information regarding the conformational changes in the protein
structure of MMOH after the binding of MMOB. MMOB has an unstructured
N-terminal peptide chain (Met1–Ser35), a well-folded core region (Asp36–Leu129)
with seven β-strands and three α-helices, and a short C-terminal sequence