Methane Monooxygenase Hydroxylase and B Component Interactions † Jingyan Zhang, Bradley J. Wallar, ‡ Codrina V. Popescu, § Daniel B. Renner, David D. Thomas, and John D. Lipscomb* Department of Biochemistry, Molecular Biology and Biophysics and Center for Metals in Biocatalysis, UniVersity of Minnesota, Minneapolis, Minnesota 55455 ReceiVed NoVember 3, 2005; ReVised Manuscript ReceiVed January 3, 2006 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 °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. Methane monooxygenase (MMO) 1 catalyzes the NADH- coupled reaction of methane with O 2 to form methanol and water (1): The soluble form of MMO (sMMO) has been isolated from several methanotrophs including Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) (2-8). sMMO consists of three protein components: a 245 kDa hydroxylase (MMOH) with a (Rγ) 2 subunit structure containing a carboxylate- and bis-µ-hydroxo-bridged dinuclear iron center in each protomer, a 38 kDa reductase (MMOR) containing † This work was supported by National Institutes of Health (NIH) Grants GM-40466 (to J.D.L.) and AR-32961 (to D.D.T.). B.J.W. was supported in part by NIH Training Grant GM-07323. C.V.P. was supported in part by NIH Postdoctoral Grant F32 GM65698. * To whom correspondence should be addressed. E-mail: lipsc001@umn.edu. Telephone: (612) 625-6454. Fax: (612) 624-5121. ‡ Current address: Department of Chemistry, Grand Valley State University, Allendale, MI 49401. § Current address: Department of Chemistry, Ursinus College, Collegeville, PA 19426. 1 Abbreviations: MMO, methane monooxygenase; sMMO, soluble form of MMO; MMOH, sMMO hydroxylase; MMOHsites, MMOH active sites (2 MMOHsites ) 1 MMOH); MMOB, sMMO component B; WT-MMOB, wild-type MMOB; MMOR, sMMO reductase; MOPS, 3-morpholinopropanesulfonic acid; O, P*, P, Q, and T, intermediates from the MMOH catalytic cycle; MMOH red , diferrous form of MMOH; MMOH ox , diferric form of MMOH; BADAN, 6-bromoacetyl-2- dimethylaminonaphthalene; 1,5-IAEDANS, 5-((((2-iodoacetyl)amino)- ethyl)amino)naphthalene-1-sulfonic acid; TCEP, tris(2-carboxyethyl)- phosphine hydrochloride; MSL spin label, 4-maleimido-2,2,6,6- tetramethyl-1-piperidinyloxy; SDSL, site-directed spin labeling; RRT, reciprocal relaxation time. NAD(P)H + H + + CH 4 + O 2 f NAD(P) + + CH 3 OH + H 2 O 2913 Biochemistry 2006, 45, 2913-2926 10.1021/bi052256t CCC: $33.50 © 2006 American Chemical Society Published on Web 02/07/2006