Abstract
Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein–hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 Å from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.
Keywords: crystal structure, iron enzyme, mechanism, oxygenase
Carboxylate-bridged diiron enzymes provide essential biological functions such as O2 transport, iron sequestration, deoxyribonucleotide synthesis, fatty acid desaturation, and hydrocarbon hydroxylation (1). All diiron hydroxylase complexes include a multisubunit hydroxylase, electron transfer proteins, and a cofactorless effector protein that is unique to the diiron hydroxylase family (2). Effector proteins are required for catalysis, and their presence is associated with improved coupling (3), shifts in redox potential (4), increased rate of catalysis (3), more efficient activation of O2 (5), and changes in regiospecificity (3, 6). Correlation of how the effector protein may induce these phenomena ultimately requires examination of high-resolution structures of the stoichiometric protein–protein complexes in multiple redox states. Previously available structures with small-molecule analogs (7, 8) or partial occupancy of an effector protein binding site (9) have provided some insight into regions of the hydroxylase that might be perturbed by these interactions. Surprisingly, however, changes in the active site were not observed, although these might reasonably be anticipated based on numerous spectroscopic studies of this enzyme family (10). Consequently, further information is needed to better understand the role of effector protein binding in catalysis by diiron hydroxylases.
Toluene 4-monooxygenase (T4moH*; 200 kDa) is composed of TmoA, TmoE, and TmoB polypeptides and has an (αβγ)2 quaternary structure (11). This enzyme hydroxylates toluene with high regiospecificity in the presence of its effector protein, T4moD (3, 12). Here, we report X-ray structures of resting T4moH, the stoichiometric complex of resting T4moH with T4moD, and the sodium dithionite-reduced complex to resolutions of 1.9, 1.9, and 1.7 Å respectively [see supporting information (SI) Table S1 for refinement statistics]. Comparison of these structures revealed changes within the active site and extending for ≈25 Å along several helices of the TmoA chain. These observations provide a striking example of the extent and coordination of structural changes arising from the function of effector proteins in diiron hydroxylase reactions.
Results and Discussion
Quaternary Structure.
Fig. 1A shows the arrangement of the TmoA, TmoE, and TmoB chains that form the T4moH (αβγ) protomer. Resting T4moH crystallized in space group P21, whereas T4moHD crystallized in space group C2221. Although resting T4moH exhibited an altered interaction between the 2 protomers in the P21 crystals, the rmsd for alignment of the backbone atoms of the αβγ protomers among the 3 structures determined was ≈0.4 Å, demonstrating equivalent arrangements of the protomers.
Fig. 1.
Structures of T4moH (gray) and T4moHD aligned by the protomer consisting of TmoA (55 kDa, blue), TmoB (10 kDa, red), and TmoE (35 kDa, green). T4moD (12 kDa) is shown in orange. (A) Overlay of protomers of T4moH and T4moHD. (B and C) Two views of resting T4moHD, with 1 protomer represented as a surface.
In the stoichiometric complex, the binding of T4moD to T4moH resulted in an overall ∼0.5-Å rmsd in the Cα positions of bound relative to unbound TmoA, minor shifts in the position of the entire TmoB chain, and no significant changes in TmoE (Fig. 1). Reduction of preformed crystals of T4moHD gave only minimal further changes in the surfaces of either T4moH or T4moD. In all structures, the surface area of the interfaces between the TmoA and TmoB (≈1,800 Å2) and the TmoA and TmoE (≈6,500 Å2) chains was extensive and largely hydrophobic. In T4moHD, the positioning of the protomers in the complex (Fig. 1 B and C) provided an additional ≈3,700 Å2 of buried surface relative to that seen in resting T4moH alone. Moreover, the interfaces between T4moD and TmoA (≈3,360 Å2) and across the protomer–protomer interface (≈860 Å2) substantially increased the buried surface area in the complex. In total, >20 new hydrogen bonds were formed in the otherwise largely hydrophobic interfaces created by T4moD binding. Of note, these include bonds between conserved Glu-214 and Gln-288 from TmoA and conserved Arg-45 and Arg-46 from the loop connecting strands β2 and β3 of T4moD. In addition, HOH6 bridged the 3.7-Å distance between Asn-202 of TmoA and Ser-82 of T4moD.
Active Site.
Fig. 2 shows 3 different structures of the T4moH active site, and Table S2 shows selected distances. Resting T4moH (Fig. 2A) contains 2 6-coordinate iron sites, virtually identical to that observed in other diiron hydroxylases (10). The diiron center, with an iron–iron distance of 3.3 Å, is coordinated by a set of glutamates and histidines that are conserved among all family members. In 1 protomer of the dimeric structure, there are 2 water or hydroxo bridges (HOH1 and HOH2) and a water molecule bound to 1 iron (HOH3). HOH4 is the terminus of a network of waters and hydrogen-bonding residues that extends ≈10 Å toward but does not reach the exterior of the TmoA chain. In the other protomer of the resting T4moH, an azide molecule replaced HOH2, but other details of the first ligation sphere are identical (Fig. S1).
Fig. 2.
Stereo images of the T4moH active site. Active-site residues and waters are labeled. Thr-201 is represented as yellow sticks, Asn-202 is represented as green sticks, and Gln-228 is represented as purple sticks throughout this work. (A) Resting T4moH. (B) Resting T4moHD. (C) Sodium dithionite-reduced T4moHD. In all diiron centers described, Glu-134 provides a bidentate bridge between the iron atoms. Additionally, Fe1 is ligated by Glu-104, His-137 ND1, and by HOH3 that is also hydrogen-bonded to Glu-104. Fe2 is ligated by Glu-197 and His-234 NE2. Glu-231 changes conformation in each structure, with the 2 resting state structures showing different monodentate coordination, while the reduced complex has a bidentate, bridging coordination. HOH4 is within hydrogen-bonding distance of the metal-coordinated carboxylates of Glu-134 and Glu-197. Distances and other images are shown in Table S2 and Fig. S1.
Fig. 2 also shows the locations of Thr-201, Asn-202, and Gln-228. This triad of residues defines a small pocket adjacent to the active site (identified in Fig. 4A by a white star). In resting T4moH (Fig. 2A), the side chains of Asn-202 and Gln-228 are exposed to solvent, and Asn-202 OD1 is hydrogen-bonded to Gln-228 N (2.8 Å). Conserved active-site residue Thr-201 has OG1 placed ≈4 Å from Glu-231 OE1 and ≈9 Å from Gln-228 NE2.
Fig. 4.
Channels and cavities found in T4moH are shown as dark surfaces. The lower panels are obtained by an ≈90° rotation from the upper panels. (A) Resting T4moH protomer showing the location of 3 channels from the exterior to the active site (orange mesh). A white star indicates a side pocket to the active site, bounded by Thr-201 (yellow sticks), Asn-202 (green sticks), Gln-228 (purple sticks), and other residues (data not shown). (B) Resting T4moHD showing residues involved in collapse of the outer part of the active-site channel (magenta mesh, Trp-89, Gln-204, Gly-207, Leu-208, Ala-210, Asp-211, Glu-214, Ala-214, Thr-281, Pro-282, Asp-285, Ser-287, Gln-288, and Phe-293), Thr-201 (yellow sticks), Asn-202 (green sticks), and Gln-228 (purple sticks). Asn-202 and Gln-228 have moved into the side pocket of 4A upon complex formation. (C) Sodium dithionite-reduced T4moHD. The comparable surface in resting T4moHD (blue mesh) is also shown. Minor changes localized near to Glu-197 and to Glu-231 were observed upon reduction.
As a consequence of T4moD binding (Fig. 2B; also see Fig. 4B), the side chains of Asn-202 and Gln-228 move ≈5 and ≈6 Å to within ≈3 Å of Thr-201 OG1, respectively, to occupy the small side pocket seen in resting T4moH. These shifts are part of a more extensive rearrangement where Gln-228 NE2 provides hydrogen bonds both to Thr-201 OG1 and HOH5, which has been newly localized in the active site. Prominently, the carboxylate group of Glu-231 has rotated ≈90° so that OE1 forms new hydrogen bonds to Thr-201 OG1 (2.7 Å) and HOH5 (2.7 Å). HOH5 is also hydrogen-bonded to HOH3. The central position of Thr-201 in stabilizing this effector protein-dependent active-site hydrogen-bonding network provides structural insight into why this residue may be conserved, but not essential, in the diiron monooxygenase family.
In T4moHD (Fig. 2B), the diiron center has 2 5-coordinate iron sites. Beyond the change in Glu-231 and appearance of HOH5, the other notable differences are the loss of μ-bridging HOH2, which yields an open coordination site on each iron atom facing into the inner active site, and presumably defines a locus for binding of O2 to initiate the catalytic cycle. A partially ordered polyethylene glycol molecule also occupies the active-site pocket, loosely bound to the metal center. There are only minor changes in the positions and interactions of the other waters and residues ligating the diiron center.
Upon reduction of T4moHD (Fig. 2C), the carboxyl group of Glu-231 rotated away from Thr-201 and HOH5 as the side chain displaced HOH1 to occupy a bridging position between the iron atoms. This bidentate bridging position increases the iron–iron distance to 3.4 Å, similar to that observed in other reduced hydroxylases in the absence of the effector protein (8, 10). Glu-197 also changed position as predicted by spectroscopic and computational studies of T4moHD (13). Other residues and waters in the active site retained their positions in the reduced complex.
Changes Away from Active Site.
In addition to active-site changes, the stoichiometric T4moHD complex revealed extensive backbone rearrangements and formation of an extended hydrogen-bonding network away from the active site (Fig. 3). A number of the residues involved in these changes are conserved throughout the hydroxylase family. The most significant deviations occurred along helices TmoA αA, αE, αF, and αH, which interact with helices α1 and α3 and strands β1, β3, β5, and β6 of T4moD. TmoA αA was affected from Asp-48 to Thr-53, as Tyr-51 adopted an unusual rotamer conformation caused by steric interactions with Ile-88 of T4moD (Fig. S2). TmoA αE was rearranged from Thr-198 to Ala-215 as the whole helix was displaced up to 2 Å in the complex (Fig. S3), and the side chains of Asn-202, Met-203 and Gln-214 were moved by close contacts with T4moD in the protein–protein interface. TmoA αF was unwound from Tyr-218 to Arg-233 (Fig. S4). Key residues in this helix altered as a consequence of effector protein binding included active-site ligand Glu-231 and outer-sphere residue Gln-228, which swung into the active site. Backbone positions of the residues in the complex were restored to that of the resting T4moH at the positionally invariant Fe2 ligand His-234. Minor structural changes within αE and αF have been observed in other hydroxylase structures (7–9), but these do not match the extent of changes found in the T4moHD complex. The greater π helical character conferred to these helices upon complex formation repositions active-site and second-sphere residues, which likely plays an important role in positioning the active site for both electron transfer and subsequent catalysis.
Fig. 3.
Changes in the TmoA chain caused by complex formation with T4moD. The TmoA chains in resting T4moH (gray) and the T4moHD complex (blue) are overlaid. The TmoA helices and other residues described in the text are labeled.
In the stoichiometric complex, TmoA αH showed rearrangements >15 residues from Phe-293 to Glu-308. The Phe-293 phenyl group changed rotamer conformation upon displacement by Met-203 from αE, the Trp-297 side chain occupied space vacated by Met-203 (Fig. S5), and Glu-303 and Arg-304 were also displaced. Some changes in αH of the apo and metal-substituted forms of MmoH have also been reported (14). It is unclear whether the changes in αH arise from interactions with the N-terminal region of T4moD or from the unwinding of αE. It is plausible that these structural changes are not induced by the N terminus of T4moD, because deletion of the N terminus up to His-10 did not significantly alter the structure of T4moD or the activity of T4moH (15). Moreover, no changes in the αH region and only minimal changes in αE were observed in the partial stoichiometry phenol hydroxylase complex, which lacks an N-terminal helix on the effector protein (9). The contributions of the N terminus in T4moD are contrasted with MmoB, where ≈30 residues of an extended N terminus unique to the methane monooxygenase subfamily plays a critical role in catalysis (16, 17). The varied contributions of the N-terminal regions of the different effector proteins to hydroxylase function suggest a subfamily-specific role for this region in catalysis.
Cavities and Channels.
Fig. 4A shows the location of 3 channels (orange mesh) that connect the exterior of resting T4moH to the active site. These channels join near TmoA residues His-96, Gln-204, and Val-276, form an intermediate chamber that lies ≈15 Å from the active site and connect to an inner chamber ≈10 Å deeper that provides part of the active site. The inner chamber has a volume of ≈250 Å3, which is comparable with the volume of the preferred substrate toluene. T4moD binding collapsed the entrance to these channels (Fig. 4B) by causing movement of 14 residues (Fig. 4B, magenta) that originally formed the channel walls. Thus, T4moD binding restricts free access between the active site and solvent. The Tyr-122 radical of ribonucleotide reductase (18), the quasi-stable peroxodiferric state of stearoyl-ACP Δ9 desaturase (19), and the peroxodiferric intermediate of Ile-100–Trp TomoH (20) are apparently stabilized by restricted access, supporting the relevance of this aspect of effector protein interactions.
In the reduced T4moHD (Fig. 4C), minor changes in the shape of the inner active site were associated with rotation of Glu-231 and shift of Glu-197 (Fig. 2C). The positions of the other active-site ligands were not changed upon reduction, and likewise Thr-201, Asn-202, Gln-228, HOH3, HOH4, HOH5, and the associated hydrogen-bonding network extending away from the active site into the TmoA helices remained as observed in resting T4moHD.
Role of Effector Protein.
Effector protein binding changes redox potentials and spectral properties (21), increases rates of steady-state and transient catalysis (5), and improves coupling (3). However, full change is often achieved with substoichiometric effector protein. For example, substoichmetric effector protein elicited complete conversion between the bound and unbound integer-spin EPR signal of reduced MmoH and maximized the regioselectivity of isopentane hydroxylation (6). In T4MO, maximal coupling and full regiospecificity were achieved at ≈0.2 T4moD per T4moH protomer, whereas in contrast steady-state turnover maximized at ≈1 T4moD per T4moH protomer (3). Thus, a long-lived conformational change in the hydroxylase has been invoked to explain the several documented effects of effector protein binding. Long-lived conformational changes have also been proposed to explain the effects of methane monooxygenase reductase on single turnover rates, redox potentials, and O2 activation (6, 22).
Other studies have shown that ligand binding can induce spectral changes and shifts in hydroxylase helices (7, 23, 24), but these previous efforts showed neither the extent nor the detail of the interaction now revealed by the stoichiometric T4moHD complex. We propose that the extensive changes in αA, αE, αF, and αH in the complex, which provide >20 new hydrogen bonds along the helices of TmoA, may allow the rearranged T4moH configuration to persist after T4moD has dissociated, thus providing a structural basis for a long-lived conformational state. The persistence of a rearranged T4moH conformation may reflect a need for 2 discrete electron transfer interactions with the electron transfer ferredoxin, T4moC, to reduce both iron atoms of a diiron center before initiation of the catalytic cycle. We propose this rearranged state is the effective configuration for binding T4moC.
In T4moH, effector protein binding closed the active-site channels and modified the cavity within the active site to a size that was comparable to the preferred substrate, toluene. This constriction of the active site may contribute to the high regiospecificity observed with T4moH (3). Gly-103 lies at the side of the inner active site away from the diiron center, and presumably near to where the methyl group of toluene will reside in the enzyme–substrate complex. The mutation Gly-103–Leu changed the regiospecificity of the T4MO reaction (3), apparently corresponding to a sterically driven shift away from para hydroxylation toward ortho hydroxylation.
Effector protein binding has been proposed to prevent adventitious reduction of the active site intermediates by blocking access of the electron transfer partner to a closest approach to the diiron center (25). When T4moD binds to T4moH, it covers all exposed residues of TmoA within 15 Å of the diiron center, supporting this proposal (Fig. S6). The steric basis for blocking of this mechanism of uncoupling may also yield the observed inhibition of steady-state catalysis (26), particularly if T4moD competes with T4moC for an overlapping binding site on T4moH.
The metal ligands and Thr residue central to the interactions observed in the T4moHD structures (Glu-197, Glu-231, and Thr-201 of T4moH) are conserved in diiron hydroxylases. A direct transfer of a proton to a peroxo intermediate or a positioning of water for this transfer have been proposed for the conserved Thr residue (27), and the participation of Thr-201 in orientation of HOH5 is consistent with the latter role in T4moH. Because mutagenesis showed that Thr-201 was not essential for steady-state catalysis (28), this residue may also participate in other steps in the reaction cycle, such as stabilization of the rearranged conformation for electron transfer.
The coordination geometry of the reduced diiron center of T4moHD is remarkably similar to that of reduced TomoH and reduced MmoH (8, 29). However, the stoichiometric T4moHD complex revealed several important alterations in second sphere residues that might also play an important role in the catalytic cycle. Of particular note is active-site water molecule HOH5 (Fig. S7), which first appears in the T4moHD complex, is retained in the reduced complex, and is coordinated by Fe1-bound water HOH3 and the newly positioned Gln-228. This assembly of well-ordered water molecules near to the diiron center may provide a proton source required for catalysis. Interestingly, the analogous residue in MmoH is Glu-241, which, if rearranged into the active site in a similar fashion, may give a substantially different pKa and ionization state for an active-site water during the chemically demanding methane hydroxylation reaction (27).
Changes in the diiron center coordination number caused by effector protein binding may also coincide with changes in redox potentials measured in MmoH upon effector protein binding (4). The present work shows that reduction can further rearrange the diiron center to a new configuration poised to bind O2 and thus initiate the oxidative steps of the reaction (13). This redox-driven rearrangement can occur in the presence of the effector protein, but may not require its presence.
The series of high-resolution structures described here provide a detailed description of the extensive protein-induced changes caused by effector protein binding in the carboxylate-bridged diiron hydroxylases. The results suggest that substrate binding is impeded when the complex is formed, implying substrate binding occurs at a different point in the catalytic cycle, perhaps before complex formation. Complex formation closes access to the active site, rearranges the diiron center ligands, and introduces open coordination sites at the diiron center facing toward the active site cavity where O2 and substrate must bind. Moreover, effector protein binding also localizes a new water molecule within hydrogen-bonding distance of these open coordination sites, possibly to serve as a source of one of the protons required for the reaction stoichiometry. The extent of hydrogen-bonding interactions away from the active site suggests that effector protein binding may produce a quasi-stable state poised for electron transfer. Reduction of the diiron center gave a further rearrangement of the ligands, and thus configured, the active site is apparently poised for rapid reaction with O2 (13).
Materials and Methods
Protein Preparation and Crystallization.
T4moH and T4moD were expressed, purified, and characterized as described (26, 30). Crystals of resting T4moH were obtained from the small scale batch method by addition of 5 μL of T4moH (≈95 μM) in 10 mM MOPS, pH 6.9, containing 200 mM NaCl to an equal volume of precipitant containing 0.1 M Hepes (pH 7.5), 15% MePEG 2K, 100 mM CaCl2, and 20 mM NaN3. Crystals grew to dimensions of ≈100 × 100 × 50 μm over ≈1 week at 22 °C. Crystals were cryoprotected by immersion in Fomblin (MW 2500) and were frozen in liquid N2.
Crystals of the T4moH and T4moD complex (T4moHD) were obtained from hanging drop vapor diffusion by addition of 2.5 μL of a solution of T4moH (140 μM) and T4moD (280 μM) in 10 mM Mops, pH 6.9, containing 50 mM NaCl to an equal volume of precipitant containing 100 mM Bis-Tris (pH 6.5), 21% (wt/vol) PEG 3350, and 200 mM Na acetate. Crystals grew to ≈200 × 200 × 200 μm after ≈1 week at 22 °C. Crystals were cryoprotected by incubation in 3% increments of PEG 3350 to a final concentration of 27% and frozen in liquid N2.
Crystals of the reduced T4moHD complex were obtained by placing crystals of resting T4moHD soaked in cryoprotectant in a solution of anaerobic cryoprotectant with 5 mM sodium dithionite and a catalytic amount of methyl viologen for ≈75 min. Crystals were then placed in liquid N2 and stored until data collection.
Structure Determination.
Diffraction data for the resting forms were collected at GMCA-CAT, beam line 23-ID-D, and diffraction data for the reduced complex were collected at LS-CAT beam line 21-ID-F at the Advanced Photon Source, Argonne National Laboratory, Argonne, IL. The data were indexed, integrated, and scaled by using HKL2000 (31). The structure of resting T4moH was solved by molecular replacement with the CCP4 suite program MOLREP (32) using 2INC as the starting model (8). The structures of the T4moHD complexes were also solved by using molecular replacement using the protomer structure of resting T4moH 3DHG as the starting model. Electron density was fit and refined by using repeated cycles of Coot (33) and REFMAC5 (34). Ramachandran and rotamer analysis were performed with Molprobity. Analysis of active site channels was done with Caver (35). Figures were prepared with PyMOL (36).
Supplementary Material
Acknowledgments.
This work was supported by National Science Foundation Grant MCB-0316232 (to B.G.F.). J.G.M. was supported by Genomic Sciences Training Program Grant 5T32HG002760. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract W-31-109-ENG-38. The General Medicine and Cancer Institutes Collaborative Access Team was supported by National Cancer Institute Grant Y1-CO-1020 and National Institute of General Medical Science Grant Y1-GM-1104.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3DHG, 3DHH, and 3DHI).
The structures referred to in this article are designated as T4MO, 4-protein toluene 4-monooxygenase complex from Pseudomonas mendocina KR1; T4moH, hydroxylase component of T4MO; T4moD, effector protein of T4MO (orange color throughout); T4moHD, stoichiometric complex of T4moH and T4moD; TmoA, 55-kDa subunit of T4moH containing the diiron center (blue color throughout); TmoB, 10-kDa subunit of T4moH (red color throughout); TmoE, 35-kDa subunit of T4moH (green color throughout); MmoH, methane monooxygenase hydroxylase; and TomoH, toluene/o-xylene monooxygenase hydroxylase.
This article contains supporting information online at www.pnas.org/cgi/content/full/0807948105/DCSupplemental.
References
- 1.Wallar BJ, Lipscomb JD. Dioxygen activation by enzymes containing binuclear nonheme iron clusters. Chem Rev. 1996;96:2625–2658. doi: 10.1021/cr9500489. [DOI] [PubMed] [Google Scholar]
- 2.Leahy JG, Batchelor PJ, Morcomb SM. Evolution of the soluble diiron monooxygenases. FEMS Microbiol Rev. 2003;27:449–479. doi: 10.1016/S0168-6445(03)00023-8. [DOI] [PubMed] [Google Scholar]
- 3.Mitchell KH, Studts JM, Fox BG. Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity. Biochemistry. 2002;41:3176–3188. doi: 10.1021/bi012036p. [DOI] [PubMed] [Google Scholar]
- 4.Paulsen KE, et al. Oxidation-reduction potentials of the methane monooxygenase hydroxylase component from methylosinus-trichosporium Ob3b. Biochemistry. 1994;33:713–722. doi: 10.1021/bi00169a013. [DOI] [PubMed] [Google Scholar]
- 5.Lee SK, Nesheim JC, Lipscomb JD. Transient intermediates of the methane monooxygenase catalytic cycle. J Biol Chem. 1993;268:21569–21577. [PubMed] [Google Scholar]
- 6.Froland WA, Andersson KK, Lee SK, Liu Y, Lipscomb JD. Methane monooxygenase component-B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions: A novel role for protein–protein interactions in an oxygenase mechanism. J Biol Chem. 1992;267:17588–17597. [PubMed] [Google Scholar]
- 7.Sazinsky MH, Lippard SJ. Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): Protein motion in the α-subunit. J Am Chem Soc. 2005;127:5814–5825. doi: 10.1021/ja044099b. [DOI] [PubMed] [Google Scholar]
- 8.McCormick MS, Sazinsky MH, Condon KL, Lippard SJ. X-ray crystal structures of manganese(II)-reconstituted and native toluene/o-xylene monooxygenase hydroxylase reveal rotamer shifts in conserved residues and an enhanced view of the protein interior. J Am Chem Soc. 2006;128:15108–15110. doi: 10.1021/ja064837r. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Sazinsky MH, Dunten PW, McCormick MS, DiDonato A, Lippard SJ. X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp OX1 phenol hydroxylase. Biochemistry. 2006;45:15392–15404. doi: 10.1021/bi0618969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Merkx M, et al. Dioxygen activation and methane hydroxylation by soluble methane monooxygenase: A tale of two irons and three proteins. Angew Chem Int Ed. 2001;40:2782–2807. doi: 10.1002/1521-3773(20010803)40:15<2782::AID-ANIE2782>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
- 11.Pikus JD, et al. Recombinant toluene 4-monooxygenase: Catalytic and Mössbauer studies of the purified diiron and Rieske components of a four-protein complex. Biochemistry. 1996;35:9106–9119. doi: 10.1021/bi960456m. [DOI] [PubMed] [Google Scholar]
- 12.Mitchell KH, Rogger CE, Gierhran T, Fox BG. Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase through the use of specifically deuterated toluene and p-xylene. Proc Natl Acad Sci USA. 2003;100:3784–3789. doi: 10.1073/pnas.0636619100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Wei P-P, Schwartz J, Mitchell K, Fox BG, Solomon E. Geometric and electronic structure studies of the binuclear nonheme ferrous active site of toluene-4-monooxygenase: Parallels with methane monooxygenase and insight into the role of the effector proteins in O2 activation. J Am Chem Soc. 2008;130:7098–7109. doi: 10.1021/ja800654d. [DOI] [PubMed] [Google Scholar]
- 14.Sazinsky MH, Merkx M, Cadieux E, Tang S, Lippard SJ. Preparation and X-ray structures of metal-free, dicobalt and dimanganese forms of soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) Biochemistry. 2004;43:16263–16276. doi: 10.1021/bi048140z. [DOI] [PubMed] [Google Scholar]
- 15.Lountos GT, Mitchell KH, Studts JM, Fox BG, Orville AM. Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein. Biochemistry. 2005;44:7131–7142. doi: 10.1021/bi047459g. [DOI] [PubMed] [Google Scholar]
- 16.Lloyd JS, Bhambra A, Murrell JC, Dalton H. Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification. Eur J Biochem. 1997;248:72–79. doi: 10.1111/j.1432-1033.1997.t01-1-00072.x. [DOI] [PubMed] [Google Scholar]
- 17.Chang SL, Wallar BJ, Lipscomb JD, Mayo KH. 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. Biochemistry. 2001;40:9539–9551. doi: 10.1021/bi0103462. [DOI] [PubMed] [Google Scholar]
- 18.Nordlund P, Eklund H. Structure and function of the Escherichia coli ribonucleotide reductase protein R2. J Mol Biol. 1993;232:123–164. doi: 10.1006/jmbi.1993.1374. [DOI] [PubMed] [Google Scholar]
- 19.Broadwater JA, Achim C, Münck E, Fox BG. Mössbauer studies of the formation and reactivity of a quasi-stable peroxodiferric intermediate of stearoyl-acyl carrier protein Δ9 desaturase. Biochemistry. 1999;38:12197–12204. doi: 10.1021/bi9914199. [DOI] [PubMed] [Google Scholar]
- 20.Murray LJ, et al. Dioxygen activation at nonheme diiron centers: Oxidation of a proximal residue in the 1100W variant of toluene/o-xylene monooxygenase hydroxylase. Biochemistry. 2007;46:14795–14809. doi: 10.1021/bi7017128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Fox BG, Liu Y, Dege JE, Lipscomb JD. Complex formation between the protein components of methane monooxygenase from Methylosinus-Trichosporium Ob3b - identification of sites of component interaction. J Biol Chem. 1991;266:540–550. [PubMed] [Google Scholar]
- 22.Blazyk JL, Gassner GT, Lippard SJ. Intermolecular electron-transfer reactions in soluble methane monooxygenase: A role for hysteresis in protein function. J Am Chem Soc. 2005;127:17364–17376. doi: 10.1021/ja0554054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Andersson KK, Elgren TE, Que L, Lipscomb JD. Accessibility to the active site of methane monooxygenase: The first demonstration of exogenous ligand binding to the diiron cluster. J Am Chem Soc. 1992;114:8711–8713. [Google Scholar]
- 24.Murray LJ, et al. Characterization of the arene-oxidizing intermediate in ToMOH as a diiron(III) species. J Am Chem Soc. 2007;129:14500–14510. doi: 10.1021/ja076121h. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Murray LJ, Lippard SJ. Substrate trafficking and dioxygen activation in bacterial multicomponent monooxygenases. Acc Chem Res. 2007;40:466–474. doi: 10.1021/ar600040e. [DOI] [PubMed] [Google Scholar]
- 26.Hemmi H, et al. Solution structure of the toluene 4-monooxygenase effector protein (T4moD) Biochemistry. 2001;40:3512–3524. doi: 10.1021/bi0013703. [DOI] [PubMed] [Google Scholar]
-
27.Lee SK, Lipscomb JD. Oxygen activation catalyzed by methane monooxygenase hydroxylase component: Proton delivery during the O
O bond cleavage steps. Biochemistry. 1999;38:4423–4432. doi: 10.1021/bi982712w. [DOI] [PubMed] [Google Scholar]
- 28.Pikus JD, et al. Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not required for catalysis. Biochemistry. 2000;39:791–799. doi: 10.1021/bi992187g. [DOI] [PubMed] [Google Scholar]
- 29.Rosenzweig AC, Nordlund P, Takahara PM, Frederick CA, Lippard SJ. Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states. Chem Biol. 1995;2:409–418. [PubMed] [Google Scholar]
- 30.Studts JM, et al. Optimized expression and purification of toluene 4-monooxygenase hydroxylase. Protein Expression Purif. 2000;20:58–65. doi: 10.1006/prep.2000.1281. [DOI] [PubMed] [Google Scholar]
- 31.Otwinowski Z, Minor W. The processing of X-ray difrraction data collected in oscillation mode. Methods Enzymol. 1997;276:307–326. doi: 10.1016/S0076-6879(97)76066-X. [DOI] [PubMed] [Google Scholar]
- 32.Vagin A, Teplyakov A. MOLREP: An automated program for molecular replacement. J Appl Crystallogr. 1997;30:1022–1025. [Google Scholar]
- 33.Emsley P, Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr D. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]
- 34.Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D. 1997;53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
- 35.Petrek M, et al. CAVER: A new tool to explore routes from protein clefts, pockets and cavities. BMC Bioinformatics. 2006;7:316. doi: 10.1186/1471-2105-7-316. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.DeLano WL. The PyMOL Molecular Graphics System. San Carlos, CA: DeLano Scientific; 2002. [Google Scholar]
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