Methanotrophs consume methane as their major carbon source and play an essential role
in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere
1–3
. These bacteria oxidize methane to methanol via soluble (sMMO) and particulate (pMMO)
methane monooxygenases
1–4
. sMMO contains three protein components, a 251 kDa hydroxylase (MMOH), a 38.6 kDa
reductase (MMOR), and a 15.9 kDa regulatory protein (MMOB) required to couple electron
consumption with substrate hydroxylation at the catalytic diiron center of MMOH
2
. Until now, the role of MMOB has remained ambiguous owing to lack of atomic-level
information about the MMOH-MMOB (hereafter H-B) complex. Here we remedy this deficiency
by providing a crystal structure of H-B, which reveals the manner by which MMOB controls
the conformation of residues in MMOH critical for substrate access to the active site.
MMOB docks at the α2β2 interface of α2β2γ2 MMOH and triggers simultaneous conformational
changes in the α-subunit that modulate O2 and CH4 access as well as proton delivery
to the diiron center. Without such careful control by MMOB of these substrate routes
to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase
5
. Biological catalysis involving small substrates is often accomplished in nature
by large proteins and protein complexes. The structure presented in this work provides
an elegant example of this principle.
Bacterial multicomponent monooxygenases (BMMs) are unique among diiron proteins by
virtue of their ability to hydroxylate a broad spectrum of hydrocarbon substrates
1–3
. Soluble methane monooxygenases (sMMOs), alkene monooxygenases (AMOs), phenol hydroxylases
(PHs), and alkene/aromatic monooxygenases (TMOs) belong to the BMM superfamily
1,2,4
. sMMO is the only BMM capable of catalyzing the conversion of methane selectively
to methanol by activation of O2 for insertion of an oxygen atom into a C–H bond (104.9
kcal/mol), as illustrated in eq 1
2,3
. The crystal
(1)
CH
4
+
O
2
+
2
H
+
+
2
e
−
→
CH
3
OH
+
H
2
O
structure of MMOH revealed a dimeric (α2β2γ2) architecture with a glutamate-bridged
diiron active site in each α-subunit
6,7
. Substrate turnover in sMMO is initiated by electron transfer from MMOR to the resting
state diiron(III) hydroxylase MMOHox, converting it to the reduced diiron(II) state,
MMOHred. In the presence of MMOB, oxygen activation at the active site of MMOHred
yields a diiron(III) peroxo intermediate that rapidly decays to form Q, the diiron(IV)
species that performs methane oxidation, returning the enzyme to the resting state
8,9
. Comparison of oxidized and reduced hydroxylase structures revealed charge neutral
active sites, reduction being accompanied by conversion of two bridging OH− ions to
water (Supplementary Fig. 1)
6,7,10
. When MMOB binds MMOH, the (FeIII)2 → (FeII)2 reduction potential is lowered, but
only in the absence of MMOR
11
. Binding of MMOB to MMOH increases the rate and specificity of substrate hydroxylation
12–14
. The nature of the internal MMOH conformational changes induced by MMOB has remained
unknown owing to the absence of structural information about the complex formed between
these two component proteins.
A crystal of H-B that diffracted to 2.9 Å resolution allowed us to determine the X-ray
structure by molecular replacement, as outlined in Methods Summary and Supplementary
Table 1. There are two H-B complexes in the asymmetric unit comprising four crystallographically
independent αβγB protomers (Supplementary Fig. 2). Within individual dimers, the protomers
are related by a non-crystallographic twofold symmetry axis (Fig. 1a and Supplementary
Fig. 3) and have nearly identical overall structures (Supplementary Table 2). MMOB
binds to the hydroxylase with its core residues (Asp 36 ~ Leu 129) located primarily
in a `canyon' region
7
formed at the α2β2 interface of the two MMOH protomers. Similar canyon motifs occur
in the hydroxylase components of phenol hydroxylase (PH) and toluene-4-monooxygenase
(T4MO) for binding their respective regulatory proteins
15–17
, but these proteins lack the N-terminal tail that is critical for the function of
sMMO (Supplementary Fig. 4). Proof that MMOB binds in the canyon of MMOH, the archetypal
and most investigated member of the BMM family, and the structure and function of
the MMOB N-terminus, are provided for the first time by the present structure determination.
NMR spectroscopic analysis
18
of unbound MMOB from M. capsulatus (Bath) revealed a compact core region (Fig. 1b)
and an unstructured N-terminal tail that is ~35 amino acids longer than the corresponding
region of regulatory proteins from all other BMM subclasses (Supplementary Fig. 4).
In the H-B complex, the MMOB core exhibits only minor structural changes with respect
to that in the unbound protein, as reflected by Cα root-mean-square-deviation values
of ~2.1 Å. The N-terminus of MMOB becomes very well ordered in H-B, forming a remarkable
ring-shaped structure on the α-subunit of MMOH (Figs. 1a and 1b). The extended N-terminus
in MMOB was previously noted to be critical for sMMO catalysis
19,20
, and those results are confirmed in the present study, wherein N-terminal truncates
(Δ 1–8, Δ 1–17, and Δ 1–33) displayed substantially reduced activity with respect
to full length MMOB (Fig. 1c).
The MMOB N-terminus binds to helices H and 4 of MMOH in the complex through hydrogen-bonding
as well as hydrophobic interactions (Supplementary Fig. 5). A small α helix (Gly 17-Phe
25) in the MMOB tail facilitates formation of its ring-shaped structure on the MMOH
surface (Fig. 1a and Supplementary Fig. 5). Within this ring structure, Phe residues
20, 24, and 25 of MMOB generate hydrophobic interactions with Lys 303 (helix H), Val
302, and Tyr 340 (helix 4 of MMOH), respectively, as shown in Supplementary Fig. 5.
These features of the N-terminus may help anchor MMOB on the MMOH surface, making
it difficult for MMOR to displace it from a preformed H-B complex. Such a consequence
would account for the diminished rate of intermolecular electron transfer observed
between MMOR and MMOH in preformed H-B
21
. In addition, hydrophilic residues including Lys 18, Asp 19, Asp 22, and Gln 23 of
MMOB, located opposite the H-B binding interface, contribute to the solubility of
the H-B complex.
Additionally, when MMOB docks onto the α-subunit of MMOH, it imparts important conformational
changes in the hydroxylase. These structural changes are largely confined to the α-subunits
and involve particularly helices E, F, H, and 4 (Supplementary Figs. 6 and 7). In
the H-B complex, Tyr 8 and Ser 111 of MMOB allosterically induce significant amino
acid side chain movements near the diiron active site in MMOH helix E. Tyr 8 forms
hydrogen bonds with Arg 307 and Glu 299 in MMOH helix H reorienting Trp 308 (Figs.
2a and 2b). This reorientation of Trp 308 is stabilized by π-interactions with Tyr
76 and Trp 78 (β3 strand) of MMOB. In addition, Ser 111 of MMOB forms a hydrogen bond
with Asn 214 in MMOH helix E, which triggers a side chain reorientation in Thr 213,
an active site, second coordination sphere residue of importance for the formation
of oxygenated intermediates in the catalytic cycle
2,3,9
and possibly proton-coupled electron transfer. In H–B, the conformational change of
Thr 213 generates hydrogen bonds with Glu 240. This event closes a pore in the MMOH
structure, the shortest access route between the diiron active site and the protein
surface defined by residues Glu 240, Thr 213 and Asn 214 (Figs 2c, d and 3). This
pore was previously proposed to be involved in proton transfer
22
.
Protons are an important substrate in BMM catalytic cycles
9,22–24
, and the H-B structure provides insight into the role that MMOB may have in facilitating
proton access to the catalytic diiron center in soluble MMO. In the H-B complex, the
conformational change of Thr 213 is accompanied by formation of a bifurcated hydrogen
bond between the hydroxyl group of this residue and the carboxylate side chain of
Glu 240. Glu 243 simultaneously undergoes a `carboxylate shift'
25
(vide infra). In the absence of MMOB, the Glu 240 and Asn 214 side chains in MMOH
are solvent accessible and linked through hydrogen bonding to a water molecule or
hydronium ion (Fig. 2c). Upon H-B complex formation (Fig. 2d), Glu 240 shifts toward
the protein interior possibly delivering a proton in the process. One possible scenario
is that, during the O2 activation steps by MMOH to form the peroxo and Q intermediates,
both of which require a proton transfer,
9,23
MMOB core binding, release, and rebinding to the hydroxylase might facilitate delivery
of the requisite two solvent-derived protons through the pore. The anchoring of MMOB
by its N-terminus may allow the core to function in this manner without complete dissociation
of the regulatory protein from the hydroxylase. The presence of these protons in the
active site would also facilitate product and hydroxide ion release during reduction
of the diiron(III) center in MMOHox to form MMOHred. Delivering protons through the
pore may be one of the primary functions of the regulatory proteins in the BMM family.
Yet another important feature of MMOB binding to the α-subunit of MMOH is to control
methane and O2 access to the active site. Previous structural analyses of MMOH crystals
soaked in solutions of halogenated substrate analogs or pressurized with Xe identified
a putative access route for these substrates
26,27
. In the MMOHox structure, cavities 2 and 3 are connected, but there is a discontinuity
between cavities 1 and 2. This break in freely diffusible space blocks access of methane
and oxygen to the active site (Fig. 3a and Supplementary Fig. 8). Molecular access
to the diiron site via the cavities is gated by residues Phe 188 and Leu 110
7,22,26
. In the H-B complex, cavities 1 and 2 become connected as a consequence of a change
in conformation of the Phe 188 side chain, and a structural comparison of MMOHox with
that of H-B highlights the difference (Fig. 3 and Supplementary Figs. 8 and 9). A
major function of MMOB binding to MMOH is, therefore, to facilitate methane and O2
access to the diiron active site by opening the gate. It is noteworthy that this structural
alteration occurs concomitantly with closure of the pore (Fig. 3 and Supplementary
Figs. 8 and 9). Opening the pore upon MMOB dissociation also supports its previously
proposed role as a hydrophilic route for methanol release
26
.
Changes also occur in the geometry of the diiron center upon MMOB binding, in accord
with spectroscopic studies that revealed conformational rearrangements of coordinated
amino acid side chains in the H-B complex
2,3,9,14
. The coordination environments of the iron atoms in H-B (Fig. 4a and Supplementary
Fig. 10) exhibit many similarities to, as well as some key differences from, those
observed in MMOHox and MMOHred (Figs. 4b and 4c and Supplementary Fig. 10)
7,10,28
. As in the other structures of MMOH, Fe1 and Fe2 in H-B are positioned within the
four-helix bundle formed by helices B, C, E, and F. The positions of helices E and
F shift upon MMOB binding, moving Fe2 ~1.1 Å from its location in MMOHox (Supplementary
Fig. 11). The coordination of Glu 243 resembles that in Hred, but the Fe⋯Fe distance
in H-B is closer to that in MMOHox.
Individual refinement of crystallographically independent diiron active sites within
the four protomers revealed the same coordinated ligands, although with slightly different
geometries (Supplementary Fig. 12). The Fe1 and Fe2 ions bond to the δ-N atoms of
His 147 and His 246, respectively, Glu 144 bridges the two metals, and Glu 209 binds
in a monodentate fashion to Fe2, all as in MMOHox structures (Fig. 4 and Supplementary
Figs. 10 and 12). The most notable change occurs in the Glu 243 side chain carboxylate,
which chelates Fe2 in a bidentate manner while being singly bonded to Fe1. In MMOHox,
the carboxylate of Glu 243 forms a single bond with Fe2, and the dangling oxygen atom
hydrogen bonds to a terminal water coordinated to Fe1 and a hydroxide ion bridging
Fe1 and Fe2. The MMOHox active site contains a water molecule terminally bound to
Fe1 that is not observed in H-B, either because it cannot be distinguished at 2.9-Å
resolution or because it is not present in H-B structure.
In conclusion, the present structure reveals how the MMOB regulatory protein controls
substrate access to the diiron center in the sMMO hydroxylase. Docking of the MMOB
core in the MMOH canyon is accompanied by ordering of its long N-terminal tail on
the α-subunit of the hydroxylase, triggering allosteric changes that control proton,
methane, and oxygen access to the active site. The timed entry of these substrates
is important to assure events required for MMOR conversion of MMOHox to MMOHred and
the generation of oxygenated intermediates that react with methane during the critical
oxygen activation and substrate hydroxylation steps of the catalytic cycle. In this
manner, MMOB can function to couple the consumption of electrons with efficient hydrocarbon
hydroxylation. Finally, the present results can be used as a leading example of the
use by nature of a large protein complex to delineate access pathways of some of its
smallest substrates to the active site of a metalloenzyme to achieve a remarkable
catalytic reaction.
Methods Summary
M. capsulatus (Bath) cultures were grown by fermentation and MMOH was purified as
described previously
9
. Recombinant full-length and truncated MMOB proteins were expressed and purified
from Escherichia coli as described
29, 30
. Crystallization, crystal structure determination, and enzyme activity studies with
the MMOB N-terminal deletion mutants were performed as described in Methods. Data
collection was performed at the Advanced Light Source beamline 8.2.2. at Lawrence
Berkeley National Laboratory and the structure was determined by molecular replacement
using the program “Phaser” with MMOHox (PDB Code: 1MTY) and MMOB (PDB Code: 1CKV)
as search models.
Methods
sMMO fermentation and purification of MMOH
M. capsulatus (Bath) cultures were fermented and MMOH was purified via DEAE-sepharose
fast-flow, S-300 size exclusion, Q sepharose, and S-200 size exclusion chromatography
9,29–32
. The final eluent was concentrated to form a pale yellow solution.
MMOB and truncated version of MMOB expression and purification
The wild type and truncated versions of MMOB were prepared recombinantly in Escherichia
coli. From a recombinant glycerol stock of native MMOB (pkk223-3-mmoB, JM105) or truncated
versions of MMOB (pET22b(+)-mmoB, BL21(DE3)), cells were grown and expressed for 3
hr at 37 °C. The native and truncated regulatory proteins were purified using Q sepharose
fast-flow and S-75 size exclusion chromatography to obtain a colorless solution
18,19,29–31
.
Enzyme activity measurement of MMOH in the presence of the full-length or N-terminal
truncated regulatory subunit (MMOB)
MMOH (1.0 μM), MMOB (2.0 μM), and MMOR (0.5 μM) were incubated with propylene in 25.0
mM phosphate buffer at pH 7.0. Steady-state kinetic data were recorded by using an
HP8452 diode array spectrophotometer
19,31
. The temperature was controlled at 45 °C with a circulating water bath. The reaction
was initiated by addition of NADH (167.0 μM) in the presence of propylene (approximately
1.0 mM). The consumption of NADH was monitored spectrophotometrically at 340 nm and
quantified by using an extinction coefficient of 6,220 M−1cm−1.
Crystallization, data collection, and structure determination
Purified MMOH (α2β2γ2) and MMOB, which was stored in 30 mM HEPES [pH7.5], 100 mM NaCl,
and 1 mM TCEP, were mixed with at a 1:2.2 molar ratio and the final concentration
was adjusted to ~10 mg/ml. Crystals were grown for one month at 18 °C by the sitting
drop vapor diffusion method in 0.1 M MES [pH 6.5] and 15% PEG 20,000 (w/v). Crystals
were flash frozen in liquid nitrogen after transferring to a cryo-protectant solution
containing the precipitant and 20% glycerol. The crystal in space group P212121, a=183.6,
b=249.0, c=122.3, gave recordable diffracted to a minimum Bragg spacing of 2.9 Å at
the Advanced Light Source (ALS) beamline 8.2.2. at Lawrence Berkeley National Laboratory.
Data were processed using XDS
33
and scaled with SCALA
34
. Molecular replacement computations with MMOHox (PDB code: 1MTY) and MMOB (PDB code:
1CKV) were performed using the program “Phaser”
35
. Model building and refinement were accomplished using Coot
36
and PHENIX.refine
37
. We generated restraints for iron atoms and ligands using the program “PHENIX” (PHENIX.metal_coordination)
37
and applied them during the refinement of the diiron center; no NCS restraints were
applied in any stage of the refinement. The final refined model contains MMOH α-subunit
(residues 15–526), β-subunit (residues 2–389) and γ-subunit (residues 3–168), and
MMOB (residues 2–133) with Rfactor and Rfree values of 20.6 and 25.8 respectively.
Supplementary Material
1