(Received for publication, November 22, 1996, and in revised form, January 2, 1997)
From the Department of Biophysics, Arrhenius
Laboratories, Stockholm University, S-106 91 Stockholm, Sweden, the
§ Department of Biochemistry, Chemical Center, Lund
University, S-221 00 Lund, Sweden, the ¶ Department of
Biochemistry, Medical School, University of Minnesota, Minneapolis,
Minnesota 55455, and the
Department of Biochemistry, University
of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway
The soluble form of methane monooxygenase (MMO)
consists of three components: reductase, hydroxylase (MMOH), and
"B" (MMOB). Resting MMOH contains a diferric
bis-µ-hydroxodinuclear iron "diamond core" cluster
which is the site of oxygen activation chemistry after reduction. Here
it is shown that -irradiation of MMOH at 77 K results in reduction
of the diiron cluster to an EPR active Fe(II)·Fe(III) mixed valence
state. At this temperature, the conformation of the enzyme remains
essentially unchanged during reduction, so the EPR-spectrum becomes a
probe of the conformation of the diferric state. The
-irradiated
MMOH exhibits EPR spectra that differ in lineshape and saturation
properties from those of the mixed valence MMOH generated by chemical
reduction in solution; annealing the
-irradiated sample at 230 K
yields the spectra of the chemically reduced sample. This demonstrates
that the conformation of diferric MMOH in the vicinity of the diiron
cluster changes during reduction to the mixed valence state. The
analogous experiment for the MMOB·MMOH complex gives a new mixed
valence species following
-irradiation that differs from all
previously reported mixed valence species. Thus, binding of MMOB also
causes a change in the conformation of diferric MMOH. It is
hypothesized that the structural changes observed for the first time
here may involve conversion of the dihydroxo-bridged diamond core
structure to one with more readily dissociable bridging oxygen ligands
to facilitate reaction with O2 following cluster
reduction.
Methane monooxygenase (MMO)1 catalyzes the chemically difficult monooxygenation of methane to form methanol (1-6).
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(Eq. 1) |
MMOH can catalyze the monooxygenation of methane and a broad range of other hydrocarbons without the presence of the other two components if the diiron cluster is nonbiologically reduced or H2O2 is supplied as a source of reduced oxygen (11-13). Nevertheless, the kinetics and other properties of the catalysis are dramatically affected by the other two components. MMOR efficiently couples the reaction to NADH oxidation (4). MMOB increases the rate of O2 reaction with the diferrous cluster by 1000-fold at 4 °C, thereby converting this step from rate-limiting to nonrate-limiting in the catalytic cycle (14) and increasing the steady state initial velocity by up to 150-fold (13, 15). These and other effects of MMOR and MMOB result from formation of a specific complexes with MMOH. There is direct evidence from CD/magnetic CD (16) and EPR (13, 15, 17-19) spectroscopic studies of the diferrous and/or mixed valence forms of the cluster that the formation of the MMOB complex causes conformational changes that affect the diiron cluster. For example, the antiferromagnetic coupling is decreased 6-fold in the mixed valence state, causing a dramatic change in the lineshape and the microwave saturation properties of the EPR spectrum when the complex is formed (15, 17).
While the effects of MMOB on the mixed valence and diferrous
states of MMOH have been extensively studied, very little information has been obtained concerning the structural consequences of MMOB binding to resting diferric MMOH. Studies using Mössbauer (17) and EXAFS (8, 20) spectroscopies have failed to detect any change in
the diiron cluster that could account for the dramatic effects of MMOB
on catalysis. Another approach to this problem is to make use of the
structurally sensitive EPR spectrum of the mixed valence state of the
enzyme to probe the resting diferric state through application of
radiolytic one-electron reduction at 77 K. Many enzymes and proteins
have been shown to harbor oxygen-bridged dinuclear iron clusters
(3-5)2; previous studies of diiron
cluster-containing proteins using this technique have revealed unique
information about the structure of the clusters in normally EPR-silent
states (21, 22). In this technique, resting MMOH is reduced in frozen
solution with mobile electrons generated by -radiolysis, as
illustrated in Scheme I.
The mixed valence diiron clusters produced at 77 K by -radiolysis
retain a conformation close to that of the original diferric clusters.
In this way, the mixed valence state trapped by the cryogenic reduction
becomes an EPR probe for the structural state of the diferric cluster.
The steric hindrance in the primary mixed valence state trapped at 77 K
in the solid matrix was found to be released by annealing the still
frozen sample at temperatures of 160-200 K for ribonucleotide
reductase R2 protein from Escherichia coli and hemerythrin
(21, 22). Here we use
-radiolytic reduction of MMOH in the solid
matrix at low temperature to show for the first time that the structure
of MMOH in the diferric form is substantially altered by both reduction
and MMOB binding.
MMOH and MMOB from Methylosinus trichosporium OB3b
were prepared as previously reported (11), and the specific activities were over 600 and 9000 milliunits/mg of protein for furan oxidation at
room temperature, respectively. Protein solutions were prepared in 50 mM Tris-HCl and 100 mM KCl buffer (pH 7.6) and
mixed with equal volumes of glycerol. The enzyme remained active in
this mixture. The final MMOH protein concentrations of the samples were
in the range 0.5-0.6 ± 2 mM MMOB. The protein
solutions were split into two identical parts, one used for chemical
reduction and the other for radiolytic one-electron reduction by
-ray irradiation at 77 K. The chemical reduction of MMOH to the
mixed valence state was achieved by incubating the oxidized protein
with 1.1 reducing equivalents of buffered sodium dithionite and
phenazine methosulfate under anaerobic conditions as described
elsewhere (11, 15). The
-irradiation of the samples was performed in
quartz tubes with internal diameters of about 3 mm immersed in liquid
nitrogen in a Dewar that was exposed to
-rays in a 137Cs
source giving a dose rate of 65 kilorads/h. EPR measurements were made
on samples that had been exposed to 2.0-2.8 megarad doses. Annealing
of the samples was performed in a cooled n-pentane bath at
230-270 K as described in the text followed by recooling to 77 K.
EPR spectra were recorded using a Bruker ESP300 X-band spectrometer
equipped with an Oxford Instruments ESR900 liquid helium cryostat.
Quantitations of the mixed valent EPR signals were done as described
previously under nonsaturating microwave power conditions (21) and by
the methods described by Aasa and Vänngård (23). The irradiation
induced strong radical signals that distorted both the base line and
the g1 values of the mixed valent iron cluster signals,
thus making the quantitations less accurate than expected for typical
EPR samples. Microwave power saturation data were obtained from the EPR
absorption derivative signal intensity (I) as a function of incident
microwave power (P). The data were analyzed using the equation, log
(I/P) = a
(b/2)(log
(P1/2
log P), from which the half
saturation power P1/2 was determined graphically (24).
Fig. 1 shows
the low temperature EPR spectra of the mixed valence forms of MMOH in
buffer/glycerol glass (1:1, by volume) generated by chemical reduction
with dithionite + phenazine methosulfate prior to freezing (trace
a) or by radiolytic reduction at 77 K (traces b and
c). Chemically reduced MMOH gives rise to a rhombic EPR
signal with g values at 1.95, 1.86, and 1.76 (11, 17, 25), which is
partially saturated under the power and temperature conditions used.
The sample of MMOH -irradiated at 77 K exhibits a complex EPR
spectrum as shown in Fig. 1, b and c. The
resonances below g = 2 are due to the mixed valence form of MMOH
induced by the
-irradiation and show significant differences from
those of the chemically reduced enzyme. The shape of the EPR spectrum (only observed below 25 K) from the mixed valence state depends on the
temperature and applied microwave power, e.g. compare Fig. 1, b and c, which were recorded at 12 and 3.8 K,
respectively. In the temperature range 6-15 K (Fig. 1 b), a
rhombic mixed valence EPR spectrum with g values at 1.94, 1.86, and
1.79 (Species I) is most intense, although at least one additional high
field resonance (g = 1.75) is apparent showing that another
species (Species II) is also present. Quantitation under nonsaturating
conditions of Species I and Species II together indicates that they
represent 22-28% of the clusters present, which is comparable with
what is observed for both ribonucleotide reductase R2 protein from E. coli and hemerythrin at similar irradiation doses (21,
22). The intensity of Species II increases with applied microwave power of up to 100 mW at 4 K (Fig. 1c). In contrast, at 4 K
Species I becomes saturated at a microwave power P
2 mW. In Fig. 1c recorded at 3.8 K and 10 mW, Species II is
therefore enhanced. The approximate lineshape of Species II
(g1 = 1.85, g2 = 1.75, and g3 = 1.70) was obtained by subtraction of the spectrum of Fig. 1b
from that of Fig. 1c to yield a null base line at g = 1.94 as shown in Fig. 1d. Approximate quantitation in the
temperature range 3.6-12 K shows that the species giving rise to
Species I represents 65 ± 20% and Species II represents 35 ± 15% of the mixed valence species. The characteristics of the
observed species are summarized in Table I.
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Although Species I seems similar to the chemically reduced mixed valence MMOH (Fig. 1a) significant differences were observed in their relaxation properties at 7 K. At that temperature, the half-microwave saturation power (P1/2) for Species I and the EPR spectrum of chemically reduced mixed valence MMOH (both measured at g = 1.86) were found to be ~20 and 0.03 mW, respectively.
These results suggest that mixed valence species of MMOH produced by
-irradiation at 77 K and by chemical means at room temperature have
distinct diiron cluster structures. Under the assumption that mixed
valence species formed at 77 K retain ligation geometry very close to
that in the original diferric center, we conclude that the resting
diferric enzyme has at least two structures, each of which is different
from the structure of the mixed valence diiron cluster produced by
chemical reduction. It is possible that the diiron sites in the
oxidized and postequilibrium mixed valence states have different
ligation geometries or even ligand structures. However, it must be
noted that EPR spectra from diiron clusters are very sensitive to small
changes in the zero field splitting parameters (D) for each
iron, the exchange coupling parameter (J), and the symmetry
of the coordination environments of each iron, so even subtle changes
in structure might cause the observed differences (21, 26-29).
The constrained Species I and II are expected to relax to an equilibrium state when the sample is warmed. Accordingly, upon annealing of the sample of mixed valence MMOH produced at 77 K for 3 min at 230 K, both EPR Species I and II disappeared and an EPR spectrum identical to that obtained for the chemically reduced mixed valence MMOH shown in Fig. 1a appeared (data not shown).3
Effect of MMOB on the Mixed Valence EPR Signals of the Iron Cluster of MMOHThe presence of MMOB affects the EPR
properties of the mixed valence state of MMOH produced either by
standard chemical reduction or by -irradiation at 77 K. As
previously observed, the chemically reduced complex of mixed valence
MMOH and MMOB (1:4) gives rise to a broad EPR signal from a single
species with g values at 1.86, 1.77, and 1.60 shown in Fig.
2a, at 3.8 K as well as at 12 K.
Fig. 2b shows the 7 K EPR spectrum (only observed
below 20 K) of the MMOH·MMOB complex (1:4) in buffer, 50% glycerol
after -irradiation at 77 K. In this spectrum, resonance features
centered at g = 1.94, 1.86, and 1.75 are clearly observed, which
are similar to those observed for Species I and II shown in Fig.
1b, albeit at significantly decreased intensity. In
addition, there are some new features that are readily observed at a
higher magnetic field. To separate out these new features, the Fig.
1b spectrum was subtracted from the spectrum shown in Fig.
2b to give a null base line at g = 1.86; this is the
spectrum shown in Fig. 2c (g1 = 1.90, g2 = 1.79, g3 = 1.59, Species III).
Quantitation under nonsaturating conditions of the mixed valent species
in Fig. 2b indicates that they represent 15-20% of
the clusters present. Species III has a different lineshape than
the chemically reduced mixed valence MMOH·MMOB complex shown in Fig.
2a. Moreover, Species III is not saturated by microwave
powers as high as 100 mW, even at 3.8 K (data not shown), whereas the
spectrum shown in Fig. 2a has a P1/2 of
approximately 15 mW at this temperature (15). Approximate quantitation
in the temperature range 3.6-12 K of the species shown in Fig.
2b suggests that Species III represents 75 ± 15% of
the mixed valent iron clusters present (see Table I).
Under our experimental conditions, all of the MMOH should be complexed with MMOB (15, 18). Indeed, saturation of the MMOH·MMOB complex is evident from the single species observed in Fig. 2a (15). Therefore, it is likely that the species similar to Species I and II, which is present in Fig. 2b, also represents a MMOB complexed form of MMOH. This suggests that the diiron cluster in about 25% of diferric MMOH is not altered significantly by MMOB.
As noted above for the 77 K -irradiated MMOH, annealing the
sample at 270 K for 2 min causes the mixed valence signals of Fig. 2,
b and c to disappear and a spectrum identical to
that of the chemically reduced mixed valence spectrum of Fig.
2a to appear (data not shown).
Taken together the results of the 77 K -irradiated reduction
studies presented here allow us to draw several conclusions about the
structure of the resting diferric cluster of MMOH and its complex with
MMOB. First, there appear to be at least two different conformers of
the diiron clusters in the uncomplexed oxidized MMOH, giving rise to
different non-equilibrium mixed valence Species I (65%) and II (35%).
The presence of two significantly different species in the diferric
state of MMOH is supported by the recent EXAFS studies (8) of the
diferric M. trichosporium OB3b MMOH in 30% glycerol, which
showed that two species in a 3:2 ratio were present with Fe-Fe
distances of 3.0 and 3.4 Å, respectively. It was proposed based on
model compound (31) and crystallographic (7, 9, 10) studies that the
populations represent Fe2(OH)2 diamond core
(3.0 Å) and single hydroxo-bridged (3.4 Å) structures, respectively.
These two structures would be expected to have different zero-field
splitting parameters and exchange coupling constants, J. Of
these two structures, the dibridged diamond core structure would be
expected to have a larger J value, and consequently a
smaller P1/2 (15, 17, 26), and therefore, it most
likely correlates with Species I. This is also consistent with the
EXAFS finding that the diamond core species was the dominant form (8),
as we observe here for Species I.
A second important finding is that both Species I and II of the non-equilibrium mixed valence state differ from the species found for chemically reduced mixed valence MMOH, showing that a conformational change occurs in the cluster during reduction. This is the first direct evidence of a structural change occurring between the diferric and the mixed valence states. The chemically reduced mixed valence state does not appear to be composed of significantly different species. Thus, it appears that one-electron reduction results in a conformational change that causes the two different subspecies of diferric MMOH to become more similar. However, any change that resulted in similar J and D values for the clusters would result in similar EPR spectra.
The third significant finding in this study is that MMOB has an effect on the MMOH diiron site in the oxidized state and that this effect is different than that previously observed for the mixed valence state (15). Again, no other technique has revealed this structural change between the oxidized and mixed valence states. It is in accord with the redox potential measurements (18) that showed that chemical reduction and MMOB binding are thermodynamically coupled, implying that some structural change occurs during reduction to the mixed valence state or when the MMOH·MMOB complex forms. The current results suggest that a change in the cluster structure is at least part of the overall changes associated with reduction to the mixed valence state and MMOB binding.
The overall effect of the binding of MMOB to MMOH seen in the
-irradiated samples is to shift the distribution of non-equilibrium mixed valence species to a form (Species III) more similar to the
minority species observed for MMOH alone (Species II). As discussed
above, this would be consistent with a shift from a predominant diamond
core structure to a mono-oxygen bridged structure, which might be the
first step in preparing the cluster to bind O2 (14).
However, EXAFS studies of the diferric MMOH and the MMOH·MMOB complex
show no change in the distribution of diamond core and mono-bridged
structures (8). Nevertheless, one electron chemical reduction of the
MMOH·MMOB complex does cause a 6-fold weaker coupling as expected for
a protonated, mono-bridged structure (15), and the crystal structure of
the diferrous state shows that one bridging oxygen from the diamond
core is lost at some stage of the reduction process (7).
One possibility is that forming the complex of diferric MMOH with MMOB
causes small structural changes that result in weakening, but not loss,
of the diamond core structure, such as a second protonation of one of
the bridging hydroxides or deprotonation of one of the cluster ligands
(Fig. 3). This weakening of the bridging ligands may
result in the conversion to a mono-bridged structure when the complex
is chemically reduced in solution or when the irradiated MMOH is
allowed to anneal. However, prior to reduction or annealing, the Fe-Fe
distances would be primarily determined by the number of bridging
oxygen ligands, and thus only minor changes in the EXAFS spectra would
be observed.
We have shown in previous studies that, of all of the effects of MMOB on MMO catalysis, the acceleration of the reaction of diferrous MMOH with O2 is the most significant. The basis for this acceleration is unknown. The results presented here show that structural changes in the MMOH diiron cluster occur as a result of formation of the MMOH·MMOB complex before reduction of MMOH to the oxygen-reactive diferrous state, thereby underscoring the potential importance of this complex in initializing catalysis.