Protein recognition in ferredoxin-P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris.
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.
Bell
Stephen G
SG
Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QR, UK. stephen.bell@chem.ox.ac.uk
Xu
Feng
F
Johnson
Eachan O D
EO
Forward
Ian M
IM
Bartlam
Mark
M
Rao
Zihe
Z
Wong
Luet-Lok
LL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D012241:Rhodopseudomonas / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
C081714:2Fe-2S ferredoxin; D001426:Bacterial Proteins; D005288:Ferredoxins; D003577:Cytochrome P-450 Enzyme System