38
2 Selective Production of Methanol …
and oxygen molecules can be enclosed are located close to the di-nuclear iron center
(see Sect. 2.2.4) [78, 79]. Phe 188 and Ile 110 act as gates between the exterior and
the interior, where the di-iron active sites are located. These conformational changes
are responsible for the effects described in points (a)–(d) above.
The binding of MMOB to MMOH also affects the conformation of MMOB.
DEER spectroscopic data suggest that the conformation of the N-terminal peptide
chain of MMOB becomes more rigid when MMOH is reduced [59]. The key role
of the MMOB N-terminus is underscored by data showing that mutants of MMOB
in which the N-terminus is disrupted display significantly reduced activity [58, 80].
The dissociation constant of the MMOH–MMOB complex was estimated by fluorescence anisotropy experiments, and MMOB was found to show a higher affinity
for MMOH red (K d = 0.17 μM) than MMOH ox (K d = 0.55 μM) [59]. The different
affinities indicate that MMOB binds to MMOH after its reduction by MMOR. The
core region of mutant MMOB with a truncated N-terminus is still able to bind to
MMOH, but with lower affinity (K d = 2.67 μM [67]). Therefore, the N-terminal
peptide chain serves to anchor MMOB to MMOH.
Based on the findings that the binding site of MMOH is shared between MMOR
and MMOB and that the affinity of MMOR and MMOB for MMOH depends on the
redox state of the di-nuclear iron center, the following functional model has been
proposed [67]:
(1) First, MMOR binds to MMOH ox by replacing MMOB; the di-nuclear iron center
of MMOH ox is then reduced by MMOR as described in Sect. 2.2.5.
(2) MMOB rebinds to the MMOH red –MMOR complex competitively to form the
MMOH red –MMOB complex. Oxygen and methane molecules can then access
the di-iron center of MMOH, as described in Sect. 2.2.4, as the binding of
MMOB to MMOH triggers simultaneous conformational changes in the αsubunit of MMOH that open the pore region to O 2 and CH 4 and enable proton
delivery to the di-iron center.
(3) After the methane oxidation reaction described in Sect. 2.2.3, MMOB dissociates from MMOH ox and the produced methanol is released.
(4) MMOR binds to MMOH ox again, initiating the catalytic cycle shown in Fig. 2.3.
This scenario is still speculative because of the absence of experimental evidence
for the displacement of MMOB by MMOR at the oxidized MMOH. However, it
is obvious that MMOB precisely controls the routes of the substrates, electrons,
and protons to the di-nuclear iron center in MMOH. Without MMOB, oxidation of
NADH to NAD
+ , rather than the oxidation of methane to methanol, is dominant in
sMMO [63].
2.3 pMMO
Particulate methane monooxygenases (pMMOs) are enzymes embedded in the membranes of methane-utilizing bacteria (methanotrophs) [1, 2, 81]. As the use of pMMOs
2 Selective Production of Methanol …
and oxygen molecules can be enclosed are located close to the di-nuclear iron center
(see Sect. 2.2.4) [78, 79]. Phe 188 and Ile 110 act as gates between the exterior and
the interior, where the di-iron active sites are located. These conformational changes
are responsible for the effects described in points (a)–(d) above.
The binding of MMOB to MMOH also affects the conformation of MMOB.
DEER spectroscopic data suggest that the conformation of the N-terminal peptide
chain of MMOB becomes more rigid when MMOH is reduced [59]. The key role
of the MMOB N-terminus is underscored by data showing that mutants of MMOB
in which the N-terminus is disrupted display significantly reduced activity [58, 80].
The dissociation constant of the MMOH–MMOB complex was estimated by fluorescence anisotropy experiments, and MMOB was found to show a higher affinity
for MMOH red (K d = 0.17 μM) than MMOH ox (K d = 0.55 μM) [59]. The different
affinities indicate that MMOB binds to MMOH after its reduction by MMOR. The
core region of mutant MMOB with a truncated N-terminus is still able to bind to
MMOH, but with lower affinity (K d = 2.67 μM [67]). Therefore, the N-terminal
peptide chain serves to anchor MMOB to MMOH.
Based on the findings that the binding site of MMOH is shared between MMOR
and MMOB and that the affinity of MMOR and MMOB for MMOH depends on the
redox state of the di-nuclear iron center, the following functional model has been
proposed [67]:
(1) First, MMOR binds to MMOH ox by replacing MMOB; the di-nuclear iron center
of MMOH ox is then reduced by MMOR as described in Sect. 2.2.5.
(2) MMOB rebinds to the MMOH red –MMOR complex competitively to form the
MMOH red –MMOB complex. Oxygen and methane molecules can then access
the di-iron center of MMOH, as described in Sect. 2.2.4, as the binding of
MMOB to MMOH triggers simultaneous conformational changes in the αsubunit of MMOH that open the pore region to O 2 and CH 4 and enable proton
delivery to the di-iron center.
(3) After the methane oxidation reaction described in Sect. 2.2.3, MMOB dissociates from MMOH ox and the produced methanol is released.
(4) MMOR binds to MMOH ox again, initiating the catalytic cycle shown in Fig. 2.3.
This scenario is still speculative because of the absence of experimental evidence
for the displacement of MMOB by MMOR at the oxidized MMOH. However, it
is obvious that MMOB precisely controls the routes of the substrates, electrons,
and protons to the di-nuclear iron center in MMOH. Without MMOB, oxidation of
NADH to NAD
+ , rather than the oxidation of methane to methanol, is dominant in
sMMO [63].
2.3 pMMO
Particulate methane monooxygenases (pMMOs) are enzymes embedded in the membranes of methane-utilizing bacteria (methanotrophs) [1, 2, 81]. As the use of pMMOs