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Article

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

Supporting Info

Matthew H. Sazinsky,^§ Pete W. Dunten, Michael S. McCormick,^§ Alberto DiDonato,^@ and Stephen J. Lippard*^§

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California 94025, and Dipartimento di Biologia Strutturale e Funzionale, Universit di Napoli Federico II and CEINGE Biotecnologie Avanzate, Napoli, Italy 80126

Biochemistry, 2006, 45 (51), pp 15392–15404

DOI: 10.1021/bi0618969

Publication Date (Web): December 2, 2006

†

This research was supported by National Institute of General Medical Sciences Grant GM32134 (S.J.L.) and the Italian Ministry of University and Research PRIN/2004 (A.D.). Portions of this study were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.

‡

The coordinates and structure factors for the PHH−PHM complex have been deposited in the Protein Data Bank for the native and SeMet enzyme as entries 2INP and 2INN, respectively.

Massachusetts Institute of Technology.

Current position: NRSA postdoctoral fellow (GM073457), Department of Biochemistry, Molecular and Cell Biology, Northwestern University, Evanston, IL 60202.

Stanford University.

Università di Napoli Federico II and CEINGE Biotecnologie Avanzate.

To whom correspondence should be addressed. E-mail: lippard@mit.edu. Telephone: (617) 253-1892. Fax: (617) 258-8150.

Abstract

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 Å resolution. PHM binds in a canyon on one side of the (αβγ)₂ PHH dimer, contacting α-subunit helices A, E, and F 12 Å 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 π-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 6 Å 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 α-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.