(Received for publication, July 14, 1995; and in revised form, August 10, 1995)
From the
Component B (MMOB) of the soluble methane monooxygenase (MMO)
system accelerates the initial velocity of methane oxidation by up to
150-fold by an unknown mechanism. The active site of MMO contains a
diferric, hydroxo-bridged diiron cluster located on the hydroxylase
component (MMOH). This cluster is reduced by the NAD(P)H-coupled
reductase component to the diferrous state, which then reacts with
O to yield two reaction cycle intermediates sequentially
termed compounds P and Q. The rate of compound P formation is shown
here to be independent of O
concentration, suggesting that
an MMOH-O
complex (compound O) is (
irreversibly)
formed before compound P. Compound Q is capable of reacting with
hydrocarbons to yield the MMOH-product complex, compound T. It is shown
here that MMOB accelerates catalysis by increasing
1000-fold the
rate of O
association and reaction with diferrous MMOH
leading to compound P. Modeling of the single turnover reaction in the
presence of substoichiometric MMOB suggests that MMOB also accelerates
the compound P to Q conversion by
40-fold. Due to this
O
-gating effect of MMOB, either compound Q or T becomes the
dominant species during turnover, depending upon the substrate
concentration and type. Because these are the species that either react
with substrate (Q) or release product (T), their buildup maximizes the
turnover rate. This is the first direct role in catalysis to be
recognized for MMOB and represents a novel method for oxygenase
regulation.
Methane monooxygenase (MMO) ()catalyzes activation of
O
for the oxidation of methane to
methanol(1, 2) .
Many other hydrocarbons serve as adventitious
substrates(3) . The soluble form of MMO isolated from Methylosinus trichosporium OB3b consists of three independent
components; a 245-kDa hydroxylase (MMOH), a 40-kDa reductase (MMOR),
and a 15-kDa protein termed component B (MMOB)(4) . The roles
of MMOR and MMOH in catalysis have been defined in a straightforward
manner. Each of the two -protomers of MMOH contains a
hydroxo-bridged diiron cluster that is essential for O
activation and hydrocarbon oxidation and thus appears to be at
the active site of the
enzyme(2, 7, 4, 5, 6, 7, 8, 9, 10, 11) .
MMOR contains FAD and a [2Fe-2S] cluster and serves to
transfer electrons from NADH to the MMOH active site
cluster(4, 12, 13) . In contrast to the well
defined roles of these components, that of MMOB has been more difficult
to determine.
MMOB contains no metals, prosthetic groups, or
cofactors. Nevertheless, in past studies, we have shown that MMOB is
active in: (i) increasing the rate of the steady state
reaction up to 150-fold(15) ; (ii) perturbing the
spectroscopic features of MMOH(15, 16) ; (iii) shifting the redox potential values of
MMOH(17) ; (iv) altering the product distribution for
complex substrates that can be hydroxylated in more than one
position(6) ; and (v) varying the rate of product
formation when MMOH functions through a HO
shunt(6, 18) . It has also been postulated that
MMOB plays roles in increasing the rate of electron transfer and in
efficiently coupling substrate oxidation to NADH
consumption(14, 19, 20, 21) .
Together, these studies suggest that MMOB alters the structure of MMOH
in a way that accelerates and potentially regulates the reaction.
However, the basis for these effects is unknown.
Recently, we have
shown that the catalytic cycle of MMOH proceeds through several
discrete intermediates(22, 23) . In an initial step,
the diferric resting state of the MMOH diiron cluster is reduced to the
diferrous state. This state reacts with O to form an
intermediate termed compound P (formally a peroxy adduct), which then
spontaneously converts to a yellow species termed compound Q. Compound
Q appears to be the species that reacts directly with substrates to
yield the terminal enzyme-product complex (compound T)(22) . In
the presence of MMOB at low substrate concentrations, these
intermediates form at progressively decreasing rates. This has allowed
some of them to be trapped and characterized (23, 24, 25) . (
)
In this study, we have investigated the effect of MMOB on the kinetics of compounds P and Q formation and decay. We show that a major new role for MMOB is to greatly increase the rates of formation of these intermediates. Our findings provide the first direct demonstration of the kinetic basis for the essential regulatory role of MMOB in establishing efficient catalysis.
Figure 1:
Effect of MMOB on the rate of
disappearance of the g = 16 EPR signal of diferrous
MMOH upon addition of O. MMOH (0.5 mM active
sites) alone (
) or in the presence of MMOB (0.5 mM)
(
) in buffer was rapidly mixed with O
-saturated
buffer (1.4 mM) (1:1) at 4 °C and then frozen at the times
indicated using the freeze-quench technique for the preparation of EPR
samples. Decay of the g = 16 EPR signal of diferrous
MMOH was fitted to a single exponential curve (solid line).
X-band EPR measurement conditions are: temperature = 8 K;
microwave power = 0.4 milliwatts.
Figure 2:
Effect of MMOB on the time course of
compound Q after reaction of diferrous MMOH with O. MMOH
(120 µM sites) and MMOB (0-240 µM) in
buffer were rapidly mixed with O
-saturated buffer (1:1) at
4 °C. The MMOB:MMOH diiron cluster ratio was as shown in the
figure. Time courses of the reactions were monitored at 430 nm, which
is maximal for compound Q (solidlines). At a ratio
of 1.3:1 MMOB:MMOH sites, the time course was essentially
superimposable on the 2:1 curve shown and is omitted here for clarity.
The dottedlines represent the simulation of the
kinetic model shown in Fig. 4.
Figure 4:
Proposed kinetic model for a single
turnover reaction in the absence of substrate. Experimentally
determined rate and dissociation constants (15, 17, 22) are shown in boldface
italics. Other constants were chosen to give the best simulation.
The abbreviations used in the model: H, diferrous
MMOH; H
, diferric MMOH; B, MMOB; O, compound O; P, compound P; Q, compound Q; H
B, OB, PB, QB, and H
B, MMOB-bound form of
each species. The free parameter values shown should be considered
approximate for the reasons given in the text. See Fig. 2and Fig. 3for simulations based on this model. Component B binding
steps were simulated under the assumption of rapid
equilibrium.
Figure 3:
Effect of MMOB on the time course of
propylene oxidation during single turnover reactions of diferrous MMOH.
Propylene oxide was extracted and analyzed after a single turnover
reaction of diferrous MMOH (0.5 mM sites) without () or
with a stoichiometric amount of MMOB (
) as described under ``Experimental Procedures.'' The solidlines indicate the simulated time courses of the product
formation based on the kinetic model shown in Fig. 4augmented
by a reaction of compound Q with propylene to form product (k = 3500 M
s
).
The simulation predicts that neither
compound Q nor compound P will accumulate in high levels in the absence
of MMOB. Accordingly, we have recently observed using
Mössbauer spectroscopy that for Fe-enriched diferrous MMOH exposed to saturating O
and frozen after 35 s at 4 °C, diferrous and diferric MMOH
were the only major species present and that these occurred in
approximately the 1:1 ratio predicted by the model for that time. (
)In contrast, in the presence of MMOB, the model predicts
that compounds P and Q will accumulate in
70% yield at different
times during the first few seconds of the reaction because their
formation rates are much faster than their decay rates. Past
Mössbauer studies have shown that these compounds
do form in high yield (23, 24, 25) at the
predicted times.
It has been shown here that MMOB plays a specific role in MMO catalysis by increasing the rate of compound P formation by as much as 1000-fold. It is likely that the rate of conversion of compound P to compound Q is also increased at least 40-fold. This has the effect of shifting the rate-limiting step in catalysis either to the reaction of substrates with compound Q to form enzyme-bound product or to the product release step, depending upon the specific substrate utilized. Together these effects maximize the rate of turnover and minimize the possibility that an activated species prior to compound Q in the catalytic cycle will react with adventitious substrates or the enzyme itself.
The physical basis for the enhancement in the reaction rate
with O remains unknown. It may be that it derives from the
lower redox potential of the MMOH-MMOB complex, which would be expected
to increase the affinity of the diiron cluster for O
and
perhaps facilitate the O-O bond-breaking chemistry (17) . (
)Alternatively, the effect may derive from an increase in
the O
accessibility of the active site or the diiron
cluster through a conformational change in accord with previous
observations demonstrating such
changes(6, 15, 17) .
We believe that the
``gating'' effect of MMOB on O reactivity
described here is its major role in catalysis. Many other effects of
MMOB documented thus far can be accounted for in the context of an
accelerated reactivity with O
that maximizes the efficiency
of catalysis. In other multicomponent oxygenases, regulation of
catalysis has been shown to be effected by factors such as: (i) specificity between components for electron transfer (see,
for example, (28) ); (ii) substrate gating of electron
transfer through coupling of redox potential to binding free
energy(29) ; and (iii) requirement for specific
component complexes for catalysis of the O
bond-breaking
reaction(30) . The regulation of catalytic efficiency through
oxygen gating appears to be a novel role for an accessory protein in an
oxygenase mechanism. A comparable role is likely to be played by the
component B from MMO systems isolated from other bacteria and perhaps
also by the similar small protein components from other, newly
recognized oxygenases that use dinuclear iron clusters in
catalysis(31) .