46
T. Yumura et al.
1 Experimental Background
Methane monooxygenase (MMO) [1–9], found in methanotroph bacteria, catalyzes
the transformation of methane and dioxygen into methanol and water at ambient pressure and temperature. Since methane is an inert hydrocarbon due to high dissociation
energy of its C–H bond (104 kcal/mol), the catalytic function of MMO has fascinated many researchers, and led them to investigate how methane is converted into
methanol under physiological condition. MMO has two different forms: cytoplasmic
soluble MMO (sMMO) [1–9] and membrane bound particulate MMO (pMMO)
[10]. To clarify mechanisms for the methane to methanol conversion by MMO, it is
important to obtain structural information of active sites responsible for the methane
hydroxylation. The first X-ray crystal structures of active sites in sMMO and pMMO
were reported in 1993 [9] and 2005 [10], respectively. The sMMO form consists of
three protein components [11–14]: a dimeric α 2 β 2 γ 2 hydroxylase (MMOH), and
NADH-dependent [Fe 2 S 2 ] and FAD-containing reductase (MMOR), and regulatory
protein (MMOB). A carboxylate-bridged diiron center is contained in each a subunit
of MMOH as the active site for the activation of dioxygen, and methane hydroxylation [9, 15–19]. On the other hand, the pMMO form is a trimer with an α 3 β 3 γ 3
polypeptide arrangement [10]. In soluble regions of each α (pmoB) subunit, mononuclear and dinuclear coppers are contained, and zinc is located within the membrane
as third metal center. The X-ray crystallographic analyses suggested that iron and
copper centers play an important role in the direct methane hydroxylation by sMMO
or pMMO, respectively [9, 10].
The catalytic cycle for sMMO has been well investigated. In a proposed catalytic
cycle (Fig. 1), the resting state of MMOH is the oxidized form (MMOH ox ), that
contains two Fe(III) cations in an octahedral coordination environment [9, 18]
(Fig. 2a). The diiron(III) state converts to the reduced Fe(II)Fe(II) state (MMOH red )
by accepting electrons provided from a reductase protein MMOR [20, 21]. During the
reduction process, a carboxylate shift of Glu243 occurs at the active site of MMOH red ,
where five-coordinate iron cations appear [18, 19] (Fig. 2b). Because of the presence
of a vacant coordination site of the diiron center of MMOH red , this intermediate interacts with dioxygen to form peroxo intermediates (MMOH peroxo ). As shown in Fig. 1,
the peroxo intermediates, which were spectroscopically monitored [22–27], subsequently converts into a bright-yellow intermediate with the high-valent Fe(IV)Fe(IV)
state [28, 29]. This intermediate, labeled Q (MMOH Q ) has a direct reactivity toward
substrate methane. Combined Mössbauer and EXAFS investigations suggested that
the active site of MMOH Q should involve a (μ-O) 2 Fe(IV) 2 diamond core (Fig. 1),
whose iron cations are antiferromagnetically coupled [30]. In addition, EXAFS analyses indicated that the coordination number of the iron cations is no greater than 5
[30].
It is important to elucidate how the (μ-O) 2 Fe(IV) 2 active site of MMOH Q oxidizes
methane into methanol. One possible mechanism for the methane hydroxylation by
MMOH is radical rebound mechanism [31], proposed by an analogy to the same
reaction by cytochrome P-450, whose active site involves a mononuclear iron-oxo
T. Yumura et al.
1 Experimental Background
Methane monooxygenase (MMO) [1–9], found in methanotroph bacteria, catalyzes
the transformation of methane and dioxygen into methanol and water at ambient pressure and temperature. Since methane is an inert hydrocarbon due to high dissociation
energy of its C–H bond (104 kcal/mol), the catalytic function of MMO has fascinated many researchers, and led them to investigate how methane is converted into
methanol under physiological condition. MMO has two different forms: cytoplasmic
soluble MMO (sMMO) [1–9] and membrane bound particulate MMO (pMMO)
[10]. To clarify mechanisms for the methane to methanol conversion by MMO, it is
important to obtain structural information of active sites responsible for the methane
hydroxylation. The first X-ray crystal structures of active sites in sMMO and pMMO
were reported in 1993 [9] and 2005 [10], respectively. The sMMO form consists of
three protein components [11–14]: a dimeric α 2 β 2 γ 2 hydroxylase (MMOH), and
NADH-dependent [Fe 2 S 2 ] and FAD-containing reductase (MMOR), and regulatory
protein (MMOB). A carboxylate-bridged diiron center is contained in each a subunit
of MMOH as the active site for the activation of dioxygen, and methane hydroxylation [9, 15–19]. On the other hand, the pMMO form is a trimer with an α 3 β 3 γ 3
polypeptide arrangement [10]. In soluble regions of each α (pmoB) subunit, mononuclear and dinuclear coppers are contained, and zinc is located within the membrane
as third metal center. The X-ray crystallographic analyses suggested that iron and
copper centers play an important role in the direct methane hydroxylation by sMMO
or pMMO, respectively [9, 10].
The catalytic cycle for sMMO has been well investigated. In a proposed catalytic
cycle (Fig. 1), the resting state of MMOH is the oxidized form (MMOH ox ), that
contains two Fe(III) cations in an octahedral coordination environment [9, 18]
(Fig. 2a). The diiron(III) state converts to the reduced Fe(II)Fe(II) state (MMOH red )
by accepting electrons provided from a reductase protein MMOR [20, 21]. During the
reduction process, a carboxylate shift of Glu243 occurs at the active site of MMOH red ,
where five-coordinate iron cations appear [18, 19] (Fig. 2b). Because of the presence
of a vacant coordination site of the diiron center of MMOH red , this intermediate interacts with dioxygen to form peroxo intermediates (MMOH peroxo ). As shown in Fig. 1,
the peroxo intermediates, which were spectroscopically monitored [22–27], subsequently converts into a bright-yellow intermediate with the high-valent Fe(IV)Fe(IV)
state [28, 29]. This intermediate, labeled Q (MMOH Q ) has a direct reactivity toward
substrate methane. Combined Mössbauer and EXAFS investigations suggested that
the active site of MMOH Q should involve a (μ-O) 2 Fe(IV) 2 diamond core (Fig. 1),
whose iron cations are antiferromagnetically coupled [30]. In addition, EXAFS analyses indicated that the coordination number of the iron cations is no greater than 5
[30].
It is important to elucidate how the (μ-O) 2 Fe(IV) 2 active site of MMOH Q oxidizes
methane into methanol. One possible mechanism for the methane hydroxylation by
MMOH is radical rebound mechanism [31], proposed by an analogy to the same
reaction by cytochrome P-450, whose active site involves a mononuclear iron-oxo