Methane Monooxygenase Hydroxylase and B Component Interactions

• DOI: 10.1021/bi052256t
• Publication Date: 2006-02-28T05:42:14Z
• Authors: Jingyan Zhang; Bradley J. Wallar; Codrina V. Popescu; Daniel B. Renner; David D. Thomas; John D. Lipscomb
• Keywords: Methane Monooxygenase; Component (thermodynamics)

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