Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris.

Journal article

CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe-2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe-S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)(-1)min(-1) for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)(-1 )min(-1) for Pux. This difference mainly arises from weak CYP199A2-PuxB binding (K (m) 34.3 vs. 0.45 microM for Pux) rather than slow electron transfer (k (cat) 19.1 vs. 37.9 s(-1) for Pux). Comparison of the 2.0-A-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin-P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe-2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.

Authors: Bell, S; Xu, F; Johnson, E; Forward, I; Bartlam, M

• Publication status: Published
• Journal: Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
• Volume: 15
• Issue: 3
• Pages: 315-328
• Publication date: 2010-03-01
• DOI: 10.1007/s00775-009-0604-7
• EISSN: 1432-1327
• ISSN: 0949-8257
• Language: English
• Keywords: Ferredoxins; Sequence Alignment; Amino Acid Sequence; Models, Molecular; Cytochrome P-450 Enzyme System; Protein Structure, Secondary; Rhodopseudomonas; Crystallography, X-Ray; Molecular Sequence Data; Mutation; Electron Transport; Bacterial Proteins