title: Hot-spot residues in the cytochrome P450cam-putidaredoxin binding interface.
abstract: Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the hydroxylation of D-camphor to produce 5-exo-hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe-2 S] cluster. Atomic-resolution structures of the Pdx-P450cam complex have recently been solved by X-ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site-directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx-P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers.
authors.affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden (The Netherlands).
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hiruma
authors.forename: Ankur
authors.initials: A
authors.lastname: Gupta
authors.forename: Alexander
authors.initials: A
authors.lastname: Kloosterman
authors.forename: Caroline
authors.initials: C
authors.lastname: Olijve
authors.forename: Betül
authors.initials: B
authors.lastname: Olmez
authors.forename: Mathias A S
authors.initials: MA
authors.lastname: Hass
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
mesh_terms: D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D005288:Ferredoxins; D011994:Recombinant Proteins; C100137:putidaredoxin; D019475:Camphor 5-Monooxygenase
keywords: NMR spectroscopy; electron transfer; kinetics; mutagenesis; protein complexes