1IWJ

Putidaredoxin-Binding Stablilizes an Active Conformer of Cytochrome P450cam in its Reduced State; Crystal Structure of Mutant(109K) Cytochrome P450cam

Summary for 1IWJ

• Entry DOI: 10.2210/pdb1iwj/pdb
• Related: 1IWI 1IWK
• Descriptor: CYTOCHROME P450-CAM, PROTOPORPHYRIN IX CONTAINING FE, CAMPHOR, ... (4 entities in total)
• Functional Keywords: putidaredoxin binding site, cytochrome p450cam, mutant 109k, riken structural genomics/proteomics initiative, rsgi, structural genomics, oxidoreductase
• Biological source: Pseudomonas putida
• Cellular location: Cytoplasm (By similarity): P00183
• Total number of polymer chains: 1
• Total formula weight: 47460.78

Authors

Nagano, S.,Shimada, H.,Tarumi, A.,Hishiki, T.,Kimata-Ariga, Y.,Egawa, T.,Park, S.-Y.,Adachi, S.,Shiro, Y.,Ishimura, Y.,RIKEN Structural Genomics/Proteomics Initiative (RSGI) (deposition date: 2002-05-15, release date: 2002-06-05, Last modification date: 2023-12-27)

Primary citation

Nagano, S.,Shimada, H.,Tarumi, A.,Hishiki, T.,Kimata-Ariga, Y.,Egawa, T.,Suematsu, M.,Park, S.-Y.,Adachi, S.,Shiro, Y.,Ishimura, Y.
Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam
Biochemistry, 42:14507-14514, 2003

PubMed Abstract: Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.

PubMed: 14661963
DOI: 10.1021/bi035410p

Experimental method

X-RAY DIFFRACTION (2 Å)