34
2 Selective Production of Methanol …
2.2.6 Electron Transfer from MMOR to MMOH
The activation of oxygen molecules by di-nuclear iron is initiated by the reduction
of the iron ions. The mechanism of electron transfer from MMOR to MMOH, which
is critical for this reduction reaction, is described in this section.
MMOR mediates the transfer of two electrons from NADH to the di-iron site in
MMOH. The electron transfer occurs via the two cofactors of MMOR: flavin adenine
dinucleotide (FAD) and the two iron/two sulfur ([2Fe–2S]) cluster [60, 61]. The protein structure of the ferredoxin domain of MMOR (residues 1–98, molecular weight
= 10,900, PDB ID 1J4Q) is known, although the overall structure of MMOR has not
been determined [62]. The reduced form of the 98-amino-acid ferredoxin domain
of MMOR was constructed using protein engineering, and its solution structure was
determined using nuclear magnetic resonance (NMR) spectroscopy and restrained
molecular dynamics calculations, as shown in Fig. 2.1. The structure consists of six
β strands arranged into two β sheets, along with three α helices. Two of these helices
form a helix−proline−helix motif, which is unprecedented among [2Fe–2S] cluster
proteins. The [2Fe–2S] cluster is coordinated by the sulfur atoms of three cysteine
residues. The domain-binding FAD accepts electrons from NADH; the domain carrying the [2Fe–2S] cofactor then accepts the electrons from the FAD domain and
donates the electrons to the di-iron center of MMOH.
MMOR plays a role in the conversion of the hydride from NADH into singleelectron equivalents and then transfers them sequentially to the di-nuclear iron center
of MMOH. This electron transfer was investigated using stopped-flow spectroscopy
[63]. According to the experimental data, NADH binds to and forms a charge-transfer
complex with the oxidized form of the FAD moiety of MMOR. Then, NADH transfers
a hydride, leading to the formation of a charge-transfer complex between the reduced
form of the FAD moiety and NAD
+ . Sequential electron transfer from the FAD moiety
to [2Fe–2S] occurs in the same kinetic phase as the release of NAD
+ from MMOR.
The intramolecular electron transfer results in the formation of a FAD semiquinone
and the reduced [2Fe–2S] state in MMOR. Thus, the [2Fe–2S] center serves to
transfer the electrons from MMOR to MMOH.
During the electron transfer, MMOR interacts with MMOH. This interaction
affects the redox potential of the di-nuclear iron center of MMOH, increasing it from
+48 to around +100 mV (vs the standard hydrogen electrode (SHE)) [64]. The positive shift makes it easier for the di-nuclear iron center of MMOH to accept electrons
from MMOR. The structure of the two-protein complex (MMOH–MMOR) has not
yet been obtained, but the formation of the complex has been investigated using various biochemical experiments and computational simulations. These investigations
have revealed that MMOR binds to the surface of MMOH in a so-called canyon-like
region, which is a polar center surrounded by hydrophobic residues (Fig. 2.9).
Some of the amino acid residues of MMOR that interact with MMOH have been
identified. Residues 24–31 in α helix 1 and residues 67–69 in α helix 2 of the ferredoxin domain of MMOR were identified via NMR spectroscopy [62]. Additionally,
the residues Glu-56 and Glu-91 of the ferredoxin domain of MMOR were identified
2 Selective Production of Methanol …
2.2.6 Electron Transfer from MMOR to MMOH
The activation of oxygen molecules by di-nuclear iron is initiated by the reduction
of the iron ions. The mechanism of electron transfer from MMOR to MMOH, which
is critical for this reduction reaction, is described in this section.
MMOR mediates the transfer of two electrons from NADH to the di-iron site in
MMOH. The electron transfer occurs via the two cofactors of MMOR: flavin adenine
dinucleotide (FAD) and the two iron/two sulfur ([2Fe–2S]) cluster [60, 61]. The protein structure of the ferredoxin domain of MMOR (residues 1–98, molecular weight
= 10,900, PDB ID 1J4Q) is known, although the overall structure of MMOR has not
been determined [62]. The reduced form of the 98-amino-acid ferredoxin domain
of MMOR was constructed using protein engineering, and its solution structure was
determined using nuclear magnetic resonance (NMR) spectroscopy and restrained
molecular dynamics calculations, as shown in Fig. 2.1. The structure consists of six
β strands arranged into two β sheets, along with three α helices. Two of these helices
form a helix−proline−helix motif, which is unprecedented among [2Fe–2S] cluster
proteins. The [2Fe–2S] cluster is coordinated by the sulfur atoms of three cysteine
residues. The domain-binding FAD accepts electrons from NADH; the domain carrying the [2Fe–2S] cofactor then accepts the electrons from the FAD domain and
donates the electrons to the di-iron center of MMOH.
MMOR plays a role in the conversion of the hydride from NADH into singleelectron equivalents and then transfers them sequentially to the di-nuclear iron center
of MMOH. This electron transfer was investigated using stopped-flow spectroscopy
[63]. According to the experimental data, NADH binds to and forms a charge-transfer
complex with the oxidized form of the FAD moiety of MMOR. Then, NADH transfers
a hydride, leading to the formation of a charge-transfer complex between the reduced
form of the FAD moiety and NAD
+ . Sequential electron transfer from the FAD moiety
to [2Fe–2S] occurs in the same kinetic phase as the release of NAD
+ from MMOR.
The intramolecular electron transfer results in the formation of a FAD semiquinone
and the reduced [2Fe–2S] state in MMOR. Thus, the [2Fe–2S] center serves to
transfer the electrons from MMOR to MMOH.
During the electron transfer, MMOR interacts with MMOH. This interaction
affects the redox potential of the di-nuclear iron center of MMOH, increasing it from
+48 to around +100 mV (vs the standard hydrogen electrode (SHE)) [64]. The positive shift makes it easier for the di-nuclear iron center of MMOH to accept electrons
from MMOR. The structure of the two-protein complex (MMOH–MMOR) has not
yet been obtained, but the formation of the complex has been investigated using various biochemical experiments and computational simulations. These investigations
have revealed that MMOR binds to the surface of MMOH in a so-called canyon-like
region, which is a polar center surrounded by hydrophobic residues (Fig. 2.9).
Some of the amino acid residues of MMOR that interact with MMOH have been
identified. Residues 24–31 in α helix 1 and residues 67–69 in α helix 2 of the ferredoxin domain of MMOR were identified via NMR spectroscopy [62]. Additionally,
the residues Glu-56 and Glu-91 of the ferredoxin domain of MMOR were identified