Methane Monooxygenase Hydroxylase and B Component Interactions

Abstract

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.

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

“…As depicted in panels E and F of Figure S1, these protein surfaces, like that in PHM, have significant hydrophobic character, but with a somewhat greater number of charged residues. NMR line broadening, mutagenesis, cross-linking, and spin-labeling studies probing interactions of MMOB with MMOH are highly consistent with a similar surface being used by MMOB to bind its hydoxylase ( Figure S5) (34,(38)(39)(40). Previously published spin-labeling experiments by our laboratory identified a different and probably incorrect MMOB binding face (41).…”

“…Previously published spin-labeling experiments by our laboratory identified a different and probably incorrect MMOB binding face (41). A recent discussion of component interactions in the sMMO system is available (40).…”

Methanobactin and the Link between Copper and Bacterial Methane Oxidation

“…There are X-ray crystal structures for the hydroxylase component (49)(50)(51)(52), nuclear magnetic resonance (NMR)-derived structures for protein B (39,53,54), and an NMR-derived structure for the flavin domain of the reductase (55). The complex formed by the three components has been studied structurally via small-angle X-ray scattering analysis and biophysically by electron paramagnetic resonance spectroscopy, ultracentrifugation, and calorimetric analysis (56,57). The catalytic cycle of sMMO has been extensively studied, and excellent progress has been made toward understanding the mechanism of oxygen and hydrocarbon activation at the binuclear iron center (45) (45,(58)(59)(60)(61)(62).…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…All these enzymes, including the soluble methane monooxygenases, contain one hydroxylase subunit with the conserved non-heme di- and increases the rates of substrate turnover [25,122,123]. Some members of the SDIMOs also contain a fourth subunit comprised of a short polypeptide with a bound Rieske center believed to be involved in electron transfer to the hydroxylase [120,[124][125][126][127].…”

“…Although these gases are produced as natural intermediates of bacterial, plant, and mammalian metabolism, the main sources of pollution is represented by natural oil seepages and oil spills [20,119]. From a biotechnological perspective, gaseous alkanes are inexpensive carbon sources for microbial and increases the rates of substrate turnover [25,122,123]. Some members of the SDIMOs also contain a fourth subunit comprised of a short polypeptide with a bound Rieske center believed to be involved in electron transfer to the hydroxylase [120,[124][125][126][127].…”