Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath).

The soluble methane monooxygenase system of Methylococcus capsulatus (Bath) includes three protein components: a 251-kDa non-heme dinuclear iron hydroxylase (MMOH), a 39-kDa iron-sulfur- and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The thermodynamic stability and kinetics of formation of complexes between oxidized MMOH and MMOB or MMOR were measured by isothermal titration calorimetry and stopped-flow fluorescence spectroscopy at temperatures ranging from 3.3 to 45 degrees C. The results, in conjunction with data from equilibrium analytical ultracentrifugation studies of MMOR and MMOB, indicate that free MMOR and MMOB exist as monomers in solution and bind MMOH with 2:1 stoichiometry. The role of component interactions in the catalytic mechanism of sMMO was investigated through simultaneous measurement of oxidase and hydroxylase activities as a function of varied protein component concentrations during steady-state turnover. The partitioning of oxidase and hydroxylase activities of sMMO is highly dependent on both the MMOR concentration and the nature of the organic substrate. In particular, NADH oxidation is significantly uncoupled from methane hydroxylation at MMOR concentrations exceeding 20% of the hydroxylase concentration but remains tightly coupled to propylene epoxidation at MMOR concentrations ranging up to the MMOH concentration. The steady-state kinetic data were fit to numerical simulations of models that include both the oxidase activities of free MMOR and of MMOH/MMOR complexes and the hydroxylase activity of MMOH/MMOB complexes. The data were well described by a model in which MMOR and MMOB bind noncompetitively at distinct interacting sites on the hydroxylase. MMOB manifests its regulatory effects by differentially accelerating intermolecular electron transfer from MMOR to MMOH containing bound substrate and product in a manner consistent with its activating and inhibitory effects on the hydroxylase.     

Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH; and a coupling protein (MMOB). MMOR uses a noncovalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), -176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-)(1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system.     

Why OrfY? Characterization of MMOD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) has been studied intensively to understand the mechanism by which it catalyzes the remarkable oxidation of methane to methanol. The cluster of genes that encode for the three characterized protein components of sMMO (MMOH, MMOB, and MMOR) contains an additional open reading frame (orfY) of unknown function. In the present study, MMOD, the protein encoded by orfY, was overexpressed as a fusion protein in Escherichia coli. Pure MMOD was obtained in high yields after proteolytic cleavage and a two-step purification procedure. Western blot analysis of Methylococcus capsulatus (Bath) soluble cell extracts showed that MMOD is expressed in the native organism although at significantly lower levels than the other sMMO proteins. The cofactorless MMOD protein is a potent inhibitor of sMMO activity and binds to the hydroxylase protein (MMOH) with an affinity similar to that of MMOB and MMOR. The addition of up to 2 MMOD per MMOH results in changes in the optical spectrum of the hydroxylase that suggest the formation of a (micro-oxo)diiron(III) center in a fraction of the MMOH-MMOD complexes. Possible functions for MMOD are discussed, including a role in the assembly of the MMOH diiron center similar to that suggested for DmpK, a protein that shares some properties with MMOD.     

Structural features of covalently cross-linked hydroxylase and reductase proteins of soluble methane monooxygenase as revealed by mass spectrometric analysis

Soluble methane monooxygenase requires complexes between its three component proteins for efficient catalysis. The hydroxylase (MMOH) must bind both to the reductase (MMOR) and to the regulatory protein (MMOB) to facilitate oxidation of methane to methanol. Although structures of MMOH, MMOB, and one domain of MMOR have been determined, less geometric information is available for the complexes. To address this deficiency, MMOH and MMOR were cross-linked by a carbodiimide reagent and analyzed by specific proteolysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and capillary high performance liquid chromatography mass spectrometry. Tandem mass spectra conclusively identified two amine-to-carboxylate cross-linked sites involving the alpha subunit of MMOH and the [2Fe-2S] domain of MMOR (MMOR-Fd). In particular, the N terminus of the MMOH alpha subunit forms cross-links to the side chains of MMOR-Fd residues Glu-56 and Glu-91. These Glu residues are close to one another on the surface of MMOR-Fd and >25 A from the [2Fe-2S] cluster. Because the N terminus of the alpha subunit of MMOH was not located in the crystal structure of MMOH, a detailed structural model of the complex based on the cross-link was precluded; however, a previously proposed binding site for MMOR on MMOH could be ruled out. Based on the cross-linking results, a MMOR E56Q/E91Q double mutant was generated. The mutant retains >80% of MMOR NADH oxidase activity but reduces sMMO activity to approximately 65% of the level supported by the wild type reductase. Cross-linking to MMOH was diminished but not abolished in the double mutant, indicating that other residues of MMOR also form cross-links to MMOH.     

Role of the C-terminal region of the B component of Methylosinus trichosporium OB3b methane monooxygenase in the regulation of oxygen activation

The effects of the C-terminal region of the B component (MMOB) of soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b on steady-state turnover, the transient kinetics of the reaction cycle, and the properties of the sMMO hydroxylase (MMOH) active site diiron cluster have been explored. MMOB is known to have many profound effects on the rate and specificity of sMMO. Past studies have revealed specific roles for the well-folded core structure of MMOB as well as the disordered N-terminal region. Here, it is shown that the disordered C-terminal region of MMOB also performs critical roles in the regulation of catalysis. Deletion mutants of MMOB missing 5, 8, and 13 C-terminal residues cause progressive decreases in the maximum steady-state turnover number, as well as lower apparent rate constants for formation of the key reaction cycle intermediate, compound Q. It is shown that this latter effect is actually due to a decrease in the rate constant for formation of an earlier intermediate, probably the hydroperoxo species, compound P. Moreover, the deletions result in substantial uncoupling at or before the P intermediate. It is proposed that this is due to competition between slow H(2)O(2) release from one of the intermediates and the reaction that carries this intermediate on to the next step in the cycle, which is slowed by the mutation. Electron paramagnetic resonance (EPR) studies of the hydroxylase component (MMOH) in the mixed valent state suggest that complexation with the mutant MMOBs alters the electronic properties of the diiron cluster in a manner distinct from that observed when wild-type MMOB is used. Active site structural changes are also suggested by a substantial decrease in the deuterium kinetic isotope effect for the reaction of Q with methane thought to be associated with a decrease in quantum tunneling in the C-H bond breaking reaction. Thus, the surface interactions between MMOH and MMOB that affect substrate oxidation and its regulation appear to require the complete MMOB C-terminal region for proper function.     

CD and MCD studies of the effects of component B variant binding on the biferrous active site of methane monooxygenase

The multicomponent soluble form of methane monooxygenase (sMMO) catalyzes the oxidation of methane through the activation of O 2 at a nonheme biferrous center in the hydroxylase component, MMOH. Reactivity is limited without binding of the sMMO effector protein, MMOB. Past studies show that mutations of specific MMOB surface residues cause large changes in the rates of individual steps in the MMOH reaction cycle. To define the structural and mechanistic bases for these observations, CD, MCD, and VTVH MCD spectroscopies coupled with ligand-field (LF) calculations are used to elucidate changes occurring near and at the MMOH biferrous cluster upon binding of MMOB and the MMOB variants. Perturbations to both the CD and MCD are observed upon binding wild-type MMOB and the MMOB variant that similarly increases O 2 reactivity. MMOB variants that do not greatly increase O 2 reactivity fail to cause one or both of these changes. LF calculations indicate that reorientation of the terminal glutamate on Fe2 reproduces the spectral perturbations in MCD. Although this structural change allows O 2 to bridge the diiron site and shifts the redox active orbitals for good overlap, it is not sufficient for enhanced O 2 reactivity of the enzyme. Binding of the T111Y-MMOB variant to MMOH induces the MCD, but not CD changes, and causes only a small increase in reactivity. Thus, both the geometric rearrangement at Fe2 (observed in MCD) coupled with a more global conformational change that may control O 2 access (probed by CD), induced by MMOB binding, are critical factors in the reactivity of sMMO.     

Preparation and X-ray structures of metal-free, dicobalt and dimanganese forms of soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath)

A three-component soluble methane monooxygenase (sMMO) enzyme system catalyzes the hydroxylation of methane to methanol at a carboxylate-bridged diiron center housed in the alpha-subunit of the hydroxylase (MMOH). Catalysis is facilitated by the presence of a regulatory protein (MMOB) and inhibited by MMOD, a protein of unknown function encoded in the sMMO operon. Both MMOB and MMOD are presumed to bind to the same region of the MMOH alpha-subunit. A colorimetric method for monitoring removal of Fe(II) from MMOH was developed using 1,10-phenanthroline and yields apo MMOH with <0.1 Fe/homodimer. With the use of this method, it was possible to investigate the X-ray structure of the apoenzyme and to perform metal reconstitution studies. Using MMOH from Methylococccus capsulatus (Bath), the effects of MMOB and MMOD on metal binding were studied and structural perturbations relevant to the function of this enzyme were identified. X-ray crystal structures of the apo, Mn(II)-soaked, and Co(II)-grown MMOH, determined to 2.3 A or greater resolution, reveal that the presence of metal ions is essential for the proper folding of helices E, F, and H of the alpha-subunit. The active sites of Mn(II)-soaked and Co(II)-grown MMOH are similar to that of reduced, native MMOH with notable differences in the metal-metal distances and ligand coordination sphere that may reflect how this dinuclear metal center might change in the presence of MMOB. MMOB and MMOD decrease the rate of removal of Fe(II) from the enzyme by 22- and 16-fold, respectively. On the basis of previous studies, it is hypothesized that MMOB, and perhaps MMOD, function to block solvent access to the MMOH active site. Finally, ITC studies and the observed disorder in helices E, F, and H in the apo and Mn(II)-soaked structures suggest that these regions of MMOH are critical for MMOB and MMOD binding.     

Residues in Methylosinus trichosporium OB3b methane monooxygenase component B involved in molecular interactions with reduced- and oxidized-hydroxylase component: a role for the N-terminus

Methane monooxygenase (MMO) is a non-heme-iron-containing enzyme which consists of 3 protein components: a hydroxylase (MMOH), an NAD(P)H-linked reductase (MMOR), and a 138-residue regulatory protein, component B (MMOB). Here, NMR spectroscopy has been used to derive interactions between MMOB and reduced and oxidized states of MMOH (245 kDa). Differential broadening of MMOB resonances in 1H-15N HSQC spectra acquired at different molar ratios of MMOH indicates interaction of both proteins, with MMOB binding more tightly to oxidized MMOH as observed previously. The most broadened backbone NH resonances suggest which residues in MMOB are part of the MMOH-binding interface, particularly when those residues are spatially close or clustered in the structure of MMOB. Although a number of different residues in MMOB appear to be involved in interacting with oxidized- and reduced-MMOH, some are identical. The two most common segments, proximal in the structure of MMOB, are beta-strand 1 with turn 1 (residues 36-46) and alpha-helix 3 going into loop 2 (residues 101-112). In addition, the N-terminus of MMOB is observed to be involved in binding to MMOH in either redox state. This is most strongly evidenced by use of a synthetic N-terminal peptide from MMOB (residues 1-29) in differential broadening 1H TOCSY studies with MMOH. Binding specificity is demonstrated by displacement of the peptide from MMOH by parent MMOB, indicating that the peptide binds in or near the normal site of N-terminal binding. The N-terminus is also observed to be functionally important. Steady-state kinetic studies show that neither a delta2-29 MMOB deletion mutant (which in fact does bind to MMOH), the N-terminal peptide, nor a combination of the two elicit the effector functions of MMOB. Furthermore, transient kinetic studies indicate that none of the intermediates of the MMOH catalytic cycle are observed if either the delta2-29 MMOB mutant or the N-terminal peptide is used in place of MMOB, suggesting that deletion of the N-terminus prevents reaction of reduced MMOH with O2 that initiates catalysis.     

An EPR study of the dinuclear iron site in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) reduced by one electron at 77 K: the effects of component interactions and the binding of small molecules to the diiron(III) center

Reduction of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) in frozen 4:1 buffer/glycerol solutions at 77 K by mobile electrons generated by gamma-irradiation produces an EPR-detectable, mixed-valent Fe(II)Fe(III) center. At this temperature the conformation of the enzyme remains essentially unaltered during reduction, so the mixed-valent EPR spectra serve to probe the active site structure of the EPR-silent, diiron(III) state. The EPR spectra of the cryoreduced samples reveal that the diiron(III) cluster of the resting hydroxylase has at least two chemically distinct forms, the structures of which differ from that of the equilibrium Fe(II)Fe(III) site. Their relative populations depend on pH, the presence of component B, and formation of the MMOH/MMOB complex by reoxidation of the reduced, diiron(II) hydroxylase. The formation of complexes between MMOB, MMOR, and the oxidized hydroxylase does not measurably affect the structure of the diiron(III) site. Cryogenic reduction in combination with EPR spectroscopy has also provided information about interaction of MMOH in the diiron(III) state with small molecules. The diiron(III) center binds methanol and phenols, whereas DMSO and methane have no measurable effect on the EPR properties of cryoreduced hydroxylase. Addition of component B favors the binding of some exogenous ligands, such as DMSO and glycerol, to the active site diiron(III) state and markedly perturbs the structure of the diiron(III) cluster complexed with methanol or phenol. The results reveal different reactivity of the Fe(III)Fe(III) and Fe(II)Fe(III) redox states of MMOH toward exogenous ligands. Moreover, unlike oxidized hydroxylase, the binding of exogenous ligands to the protein in the mixed-valent state is allosterically inhibited by MMOB. The differential reactivity of the hydroxylase in its diiron(III) and mixed-valent states toward small molecules, as well as the structural basis for the regulatory effects of component B, is interpreted in terms of a model involving carboxylate shifts of a flexible glutamate ligand at the Fe(II)Fe(III) center.     

Positively charged amino acids are essential for electron transfer and protein-protein interactions in the soluble methane monooxygenase complex from Methylococcus capsulatus (Bath)

The soluble methane monooxygenase (sMMO) complex from Methylococcus capsulatus (Bath) catalyses oxygen- and NAD(P)H-dependent oxygenation of methane, propene, and other substrates. Whole-complex sMMO oxygenase activity requires all three sMMO components: the hydroxylase, the reductase, and protein B. Also, in the presence of hydrogen peroxide, the hydroxylase alone catalyzes substrate oxygenation via the peroxide shunt reaction. We investigated the effect of amine cross-linking on hydroxylase activity to probe the role of a gross conformational change that occurs in the hydroxylase upon binding of the other protein components. The cross-linker inhibited hydroxylase activity in the whole complex, but this effect was due to covalent modification of primary amine groups rather than cross-linking. Covalent modification of arginine side-chains on the hydroxylase had a similar effect, but, most remarkably, neither form of modification affected the activity of the hydroxylase via the peroxide shunt reaction. It was shown that covalent modification of positively charged groups on the hydroxylase, which occurred at multiple sites, interfered with its physical and functional interactions with protein B and with the passage of electrons from the reductase. These results indicate that protein B and the reductase of the sMMO complex interact via positively charged groups on the surface of the hydroxylase to induce a conformational change that is necessary for delivery of electrons into the active site of the hydroxylase. Modification of positively charged groups on protein B had no effect on its function, consistent with the hypothesis that positively charged groups on the hydroxylase interact with negative charges on protein B. Thus, we have discovered a means of specifically inactivating the interactions between the sMMO complex while preserving the catalytic activity of the hydroxylase active site which provides a new method of studying intercomponent interactions within sMMO.     

Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B

The regulatory component MMOB of soluble methane monooxygenase (sMMO) has been hypothesized to control access of substrates into the active site of the hydroxylase component (MMOH) through formation of a size specific channel or region of increased structural flexibility tuned to methane and O(2). Accordingly, a decrease in the size of four MMOB residues (N107G/S109A/S110A/T111A, the Quad mutant) was shown to accelerate the reaction of substrates larger than methane with the reactive MMOH intermediate Q [Wallar, B. J., and Lipscomb, J. D. (2001) Biochemistry 40, 2220-2233]. Here, this hypothesis is tested by construction of single and double mutations involving the residues of the Quad mutant. It is shown that mutations of residues that extend into the core structure of MMOB alter many aspects of the MMOH catalyzed reaction but do not mimic the effects of the Quad mutant. In contrast, the MMOB residues that are thought to form part of the interface in the MMOH-MMOB complex increase active site accessibility as observed for the Quad mutant. In particular, the mutant T111A mimics most of the effects of the Quad mutant; thus, Thr111 is proposed to most directly control access. Unexpectedly, mutation of Thr111 to the larger Tyr greatly increases the rate constant for the reaction of larger substrates such as ethane, furan, and nitrobenzene with Q while decreasing the rate constant for the reaction with methane. Other steps in the cycle are dramatically slowed, the regiospecificity for nitrobenzene oxidation is altered, and 10-fold more T111Y than wild-type MMOB is required to maximize the rate of turnover. Thus, T111Y appears to make a more extensive change in local interface structure that allows hydrocarbons at least as large as ethane to bind and react with Q similarly. As a result, the bond cleavage rates for methane, ethane, and their deuterated analogues are shown for the first time to correlate with bond strength in accord with a mechanism in which C-H bond cleavage occurs during reaction of substrates with Q.     

Regulation of methane monooxygenase catalysis based on size exclusion and quantum tunneling

The hydroxylase component (MMOH) of the soluble form of methane monooxygenase (sMMO) isolated from Methylosinus trichosporium OB3b catalyzes both the O2 activation and the CH4 oxidation reactions at the oxygen-bridged dinuclear iron cluster present in its buried active site. During the reaction cycle, the diiron cluster forms a bis-mu-oxo-(Fe(IV))2 intermediate termed compound Q (Q) that reacts directly with methane. Many adventitious substrates also react with Q, most at a relatively slow rate. We have proposed that Q reacts preferentially with CH4 because the sMMO regulatory component MMOB induces a size selective pore into the MMOH active site as the two components form a complex. Support for this proposal has come through the observation of a nonlinear Arrhenius plot for the CH4 oxidation, presumably due to a shift in rate-limiting step from substrate binding at low temperature to C-H bond cleavage at high temperature. Reactions of all substrates other than CH4 fail to exhibit a break in the Arrhenius plot because binding is always rate limiting in the temperature range explored. Here we show that it is possible to induce a break in the Arrhenius plot for the ethane reaction with Q by using an MMOB mutant termed DBL2 (S109A/T111A) in which residues at the MMOH-MMOB interface are reduced in size. We hypothesize that this increases the ethane binding rate and shifts the Arrhenius breakpoint into the observable temperature range. As a result of this shift, the kinetic and activation parameters of the C-H bond breaking reaction for both methane and ethane can be observed using the DBL2 mutant. A 2H-KIE is observed for both substrate oxidation reactions when using DBL2, whereas only CH4 oxidation exhibits an effect when using wild type MMOB, consistent with the C-H bond cleaving reaction becoming at least partially rate limiting for ethane. Analysis of the temperature dependence of the 2H-KIE for ethane and methane for reactions using both mutant and wild type forms of MMOB suggests that quantum tunneling plays a significant role in methane oxidation but not ethane oxidation.     

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.     

X-ray absorption spectroscopic studies of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b

The conversion from methane to methanol is catalyzed by methane monooxygenase (MMO) in methanotrophic bacteria. Earlier work on the crystal structures of the MMO hydroxylase component (MMOH) from Methylococcus capsulatus (Bath) at 4??°C and –160??°C has revealed two different core arrangements for the diiron active site. To ascertain the generality of these results, we have now carried out the first structural characterization on MMOH from Methylosinus trichosporium OB3b. Our X-ray absorption spectroscopic (XAS) analysis suggests the presence of two Fe-Fe distances of about 3?Å and 3.4?Å, which are proposed to reflect two populations of MMOH molecules with either a bis(μ-hydroxo)(μ-carboxylato)- or a (μ-hydroxo)(μ-carboxylato)diiron(III) core structure, respectively. The observation of these two different core structures, together with the crystallographic results of the MMOH from Methylococcus capsulatus (Bath), suggests the presence of an equilibrium that may reflect a core flexibility that is required to accommodate the various intermediates in the catalytic cycle of the enzyme. XAS studies on the binding of component B (MMOB) to the hydroxylase component show that MMOB does not perturb either this equilibrium or the gross structure of the oxidized diiron site in MMOH.     

Methane monooxygenase hydroxylase and B component interactions

The interaction of the soluble methane monooxygenase regulatory component (MMOB) and the active site-bearing hydroxylase component (MMOH) is investigated using spin and fluorescent probes. MMOB from Methylosinus trichosporium OB3b is devoid of cysteine. Consequently, site-directed mutagenesis was used to incorporate single cysteine residues, allowing specific placement of the probe molecules. Sixteen MMOB Cys mutants were prepared and labeled with the EPR spin probe 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (MSL). Spectral evaluation of probe mobility and accessibility to the hydrophilic spin-relaxing agent NiEDDA showed that both properties decrease dramatically for a subset of the spin labels as the complex with MMOH forms, thereby defining the likely interaction surface on MMOB. This surface contains MMOB residue T111 thought to play a role in substrate access into the MMOH active site. The surface also contains several hydrophilic residues and is ringed by charged residues. The surface of MMOB opposite the proposed binding surface is highly charged, consistent with solvent exposure. Probes of both of the disordered N- and C-terminal regions remain highly mobile and exposed to solvent in the MMOH complex. Spin-labeling studies show that residue A62 of MMOB is located in a position where it can be used to monitor MMOH-MMOB complex formation without perturbing the process. Accordingly, steady-state kinetic assays show that it can be changed to Cys (A62C) and labeled with the fluorescent probes 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) or 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) without loss of the ability of MMOB to promote turnover. The BADAN fluorescence is partially quenched and red shifted as the complex with MMOH forms, allowing affinity measurements. It is shown that the high affinity of labeled MMOB (K(D) = 13.5 nM at pH 6.6, 25 degrees C) for the oxidized MMOH decreases substantially with increasing pH and increasing ionic strength but is nearly unaffected by addition of nonionic detergents. Similarly, the fluorescence anisotropy of the 1,5-IAEDANS-labeled A62C-MMOH complex is perturbed by salts but not nonionic detergents. This suggests that the MMOB-MMOH complex is stabilized by electrostatic interactions consistent with the characteristics of the proposed binding surface. Reduction of MMOH results in a 2-3 order of magnitude decrease in the affinity of the BADAN-labeled A62C-MMOB-MMOH complex, consistent with previous indications of structural change associated with reduction of the active site dinuclear iron cluster. Utilizing BADAN-labeled MMOB, the association and dissociation rate constants for the MMOB-MMOH binding reaction were determined and found to be consistent with a two-step process, possibly involving rapid association followed by a slower conformational change. The latter may be related to the regulation of substrate access into the active site of MMOH.     

Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) catalyzes the selective oxidation of methane to methanol, the first step in the primary catabolic pathway of methanotrophic bacteria. A reductase (MMOR) mediates electron transfer from NADH through its FAD and [2Fe-2S] cofactors to the dinuclear non-heme iron sites housed in a hydroxylase (MMOH). The structurally distinct [2Fe-2S], FAD, and NADH binding domains of MMOR facilitated division of the protein into its functional ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) component domains. The 10.9 kDa MMOR-Fd (MMOR residues 1-98) and 27.6 kDa MMOR-FAD (MMOR residues 99-348) were expressed and purified from recombinant Escherichia coli systems. The Fd and FAD domains have absorbance spectral features identical to those of the [2Fe-2S] and flavin components, respectively, of MMOR. Redox potentials, determined by reductive titrations that included indicator dyes, for the [2Fe-2S] and FAD cofactors in the domains are as follows: -205.2 +/- 1.3 mV for [2Fe-2S](ox/red), -172.4 +/- 2.0 mV for FAD(ox/sq), and -266.4 +/- 3.5 mV for FAD(sq/hq). Kinetic and spectral properties of intermediates observed in the reaction of oxidized MMOR-FAD (FAD(ox)) with NADH at 4 degrees C were established with stopped-flow UV-visible spectroscopy. Analysis of the influence of pH on MMOR-FAD optical spectra, redox potentials, and NADH reaction kinetics afforded pK(a) values for the semiquinone (FAD(sq)) and hydroquinone (FAD(hq)) MMOR-FAD species and two protonatable groups near the flavin cofactor. Electron transfer from MMOR-FAD(hq) to oxidized MMOR-Fd is extremely slow (k = 1500 M(-1) s(-1) at 25 degrees C, compared to 90 s(-1) at 4 degrees C for internal electron transfer between cofactors in MMOR), indicating that cofactor proximity is essential for efficient interdomain electron transfer.     

Adventitious reactions of alkene monooxygenase reveal common reaction pathways and component interactions among bacterial hydrocarbon oxygenases

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 belongs to a family of multicomponent nonheme binuclear iron-centre oxygenases that includes the soluble methane monooxygenases (sMMOs) found in some methane-oxidizing bacteria. The enzymes catalyse the insertion of oxygen into organic substrates (mostly hydrocarbons) at the expense of O2 and NAD(P)H. AMO is remarkable in its ability to oxidize low molecular-mass alkenes to their corresponding epoxides with high enantiomeric excess. sMMO and other well-characterized homologues of AMO exhibit two adventitious activities: (1) turnover-dependent inhibition by alkynes and (2) activation by hydrogen peroxide in lieu of oxygen and NAD(P)H (the peroxide shunt reaction). Previous studies of the AMO had failed to detect these activities and opened the possibility that the mechanism of AMO might be fundamentally different from that of its homologues. Thanks to improvements in the protocols for cultivation of R. rhodochrous B-276 and purification and assay of AMO, it has been possible to detect and characterize turnover-dependent inhibition of AMO by propyne and ethyne and activation of the enzyme by hydrogen peroxide. These results indicate a similar mechanism to that found in sMMO and also, unexpectedly, that the enantiomeric excess of the chiral epoxypropane product is significantly reduced during the peroxide shunt reaction. Inhibition of the oxygen/NADH-activated reaction, but not the peroxide shunt, by covalent modification of positively charged groups revealed an additional similarity to sMMO and may indicate very similar patterns of intersubunit interactions and/or electron transfer in both enzyme complexes.     

Solution structure of component B from methane monooxygenase derived through heteronuclear NMR and molecular modeling

Methane monooxygenase (MMO) is a nonheme iron-containing enzyme which consists of three protein components: a hydroxylase (MMOH), an NADH-linked reductase (MMOR), and a small "B" component (MMOB) which plays a regulatory role. Here, 1H, 13C, 15N heteronuclear 2D and 3D NMR spectroscopy has been used to derive the solution structure of the 138 amino acid MMOB protein in the monomer state. Pulse field gradient NMR self-diffusion measurements indicate predominant formation of dimers at 1 mM MMOB and monomers at or below 0.2 mM. MMOB is active as a monomer. Aggregate exchange broadening and limited solubility dictated that multidimensional heteronuclear NMR experiments had to be performed at a protein concentration of 0.2 mM. Using 1340 experimental constraints (1182 NOEs, 98 dihedrals, and 60 hydrogen bonding) within the well-folded part of the protein (residues 36-126), MMOB structural modeling produced a well-defined, compact alpha/beta fold which consists of three alpha-helices and six antiparallel beta-strands arranged in two domains: a betaalphabetabeta and a betaalphaalphabetabeta. Excluding the ill-defined N- and C-terminal segments (residues 1-35 and 127-138), RMS deviations are 1.1 A for backbone atoms and 1.6 A for all non-hydrogen atoms. Compared to the lower resolution NMR structure for the homologous protein P2 from the Pseudomonas sp. CF600 phenol hydroxylase system (RMSD = 2.48 A for backbone atoms) (Qian, H., Edlund, U., Powlowski, J., Shingler, V., and Sethson, I. (1997) Biochemistry, 36, 495-504), that of MMOB reveals a considerably more compact protein. In particular, MMOB lacks the large "doughnut" shaped cavity reported for the P2 protein. This difference may result from the limited number of long-range NOEs that were available for use in the modeling of the P2 structure. This NMR-derived structure of MMOB, therefore, presents the first high-resolution structure of a small protein effector of a nonheme oxygenase system.     

Methane monooxygenase component B mutants alter the kinetics of steps throughout the catalytic cycle

Component interactions play important roles in the regulation of catalysis by methane monooxygenase (MMO). The binding of component B (MMOB) to the hydroxylase component (MMOH) has been shown in previous studies to cause structural changes in MMOH that result in altered thermodynamic and kinetic properties during the reduction and oxygen binding steps of the catalytic cycle. Here, specific amino acid residues of MMOB that play important roles in the interconversion of several intermediates of the MMO cycle have been identified. Both of the histidine residues in Methylosinus trichosporium OB3b MMOB (H5 and H33) were chemically modified by diethylpyrocarbonate (DEPC). Although the DEPC--MMOB species exhibited only minor changes relative to unmodified MMOB in steady-state MMO turnover, large decreases in the formation rate constants of the reaction cycle intermediates, compound P and compound Q, were observed. The site specific mutants H5A, H33A, and H5A/H33A were made and characterized. H5A and wild type MMOB elicited similar steady-state and transient kinetics, although the mutant caused a slightly lower rate constant for Q formation. Conversely, H33A exhibited a >50-fold decrease in the P formation rate constant, which resulted in slower formation of Q. The kinetics of the double mutant (H5A/H33A) were similar to those of H33A, suggesting that the highly conserved residue, H33, has the most significant effect on the efficient progress of the cycle. Ongoing NMR investigations of residues perturbed by formation of the MMOH-MMOB complex suggested construction of the MMOB N107G/S109A/S110A/T111A quadruple mutant. This mutant was found to elicit a nearly 2-fold increase in specific activity for steady-state MMO turnover of large substrates such as furan and nitrobenzene but caused no similar increase for the physiological substrate, methane. While the quadruple mutant did not have a significant effect on P and Q formation, it caused an almost 3-fold increase in the decay rate constant of Q for furan oxidation and a 2-fold faster product release rate constant for p-nitrophenol resulting from nitrobenzene oxidation. Conversely, this mutant caused the Q decay rate constant to decrease 7-fold for methane oxidation but left the product release step unaffected. These results show for the first time that MMOB exerts influence at late as well as early steps in the catalytic cycle. They also suggest that MMOB plays a critical role in determining the ability of MMO to distinguish between methane and larger substrates.     

X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase

Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.