PDBsum entry 1p0x

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Oxidoreductase PDB id
Protein chains
441 a.a. *
HEM ×2
Waters ×1238
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: F393y mutant heme domain of flavocytochrome p450 bm3
Structure: Bifunctional p-450:nadph-p450 reductase. Chain: a, b. Fragment: heme domain, residues 1-455 of sws p14779. Synonym: heme domain of flavocytochrome p450 bm3. Cytochrome p450(bm-3). P450bm-3. Engineered: yes. Mutation: yes
Source: Bacillus megaterium. Organism_taxid: 1404. Gene: cyp102a21. Expressed in: escherichia coli. Expression_system_taxid: 562.
2.00Å     R-factor:   0.172     R-free:   0.237
Authors: T.W.B.Ost,J.Clark,C.S.Miles,M.D.Walkinshaw,G.A.Reid, S.K.Chapman,S.Daff,C.G.Mowat
Key ref: T.W.Ost et al. (2003). Oxygen activation and electron transfer in flavocytochrome P450 BM3. J Am Chem Soc, 125, 15010-15020. PubMed id: 14653735 DOI: 10.1021/ja035731o
11-Apr-03     Release date:   09-Dec-03    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P14779  (CPXB_BACME) -  Bifunctional P-450/NADPH-P450 reductase
1049 a.a.
441 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class 2: E.C.  - Unspecific monooxygenase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: RH + reduced flavoprotein + O2 = ROH + oxidized flavoprotein + H2O
+ reduced flavoprotein
+ O(2)
+ oxidized flavoprotein
+ H(2)O
      Cofactor: Heme-thiolate
   Enzyme class 3: E.C.  - NADPH--hemoprotein reductase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: NADPH + n oxidized hemoprotein = NADP+ + n reduced hemoprotein
+ n oxidized hemoprotein
= NADP(+)
+ n reduced hemoprotein
      Cofactor: FAD; FMN
Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     oxidation-reduction process   1 term 
  Biochemical function     oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen     3 terms  


DOI no: 10.1021/ja035731o J Am Chem Soc 125:15010-15020 (2003)
PubMed id: 14653735  
Oxygen activation and electron transfer in flavocytochrome P450 BM3.
T.W.Ost, J.Clark, C.G.Mowat, C.S.Miles, M.D.Walkinshaw, G.A.Reid, S.K.Chapman, S.Daff.
In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s(-)(1). The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.

Literature references that cite this PDB file's key reference

  PubMed id Reference
21110374 C.J.Whitehouse, W.Yang, J.A.Yorke, B.C.Rowlatt, A.J.Strong, C.F.Blanford, S.G.Bell, M.Bartlam, L.L.Wong, and Z.Rao (2010).
Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3).
  Chembiochem, 11, 2549-2556.
PDB code: 3m4v
20180779 H.M.Girvan, C.W.Levy, P.Williams, K.Fisher, M.R.Cheesman, S.E.Rigby, D.Leys, and A.W.Munro (2010).
Glutamate-haem ester bond formation is disfavoured in flavocytochrome P450 BM3: characterization of glutamate substitution mutants at the haem site of P450 BM3.
  Biochem J, 427, 455-466.
PDB codes: 3kx3 3kx4 3kx5
20446763 T.C.Pochapsky, S.Kazanis, and M.Dang (2010).
Conformational plasticity and structure/function relationships in cytochromes P450.
  Antioxid Redox Signal, 13, 1273-1296.  
19492389 C.J.Whitehouse, S.G.Bell, W.Yang, J.A.Yorke, C.F.Blanford, A.J.Strong, E.J.Morse, M.Bartlam, Z.Rao, and L.L.Wong (2009).
A highly active single-mutation variant of P450BM3 (CYP102A1).
  Chembiochem, 10, 1654-1656.
PDB code: 3hf2
18815130 J.Tejero, A.Biswas, Z.Q.Wang, R.C.Page, M.M.Haque, C.Hemann, J.L.Zweier, S.Misra, and D.J.Stuehr (2008).
Stabilization and Characterization of a Heme-Oxy Reaction Intermediate in Inducible Nitric-oxide Synthase.
  J Biol Chem, 283, 33498-33507.
PDB code: 3dwj
18161730 M.J.Cryle, and J.J.De Voss (2008).
The role of the conserved threonine in P450 BM3 oxygen activation: substrate-determined hydroxylation activity of the Thr268Ala mutant.
  Chembiochem, 9, 261-266.  
18473391 R.J.Branco, A.Seifert, M.Budde, V.B.Urlacher, M.J.Ramos, and J.Pleiss (2008).
Anchoring effects in a wide binding pocket: the molecular basis of regioselectivity in engineered cytochrome P450 monooxygenase from B. megaterium.
  Proteins, 73, 597-607.  
17650504 I.G.Denisov, Y.V.Grinkova, M.A.McLean, and S.G.Sligar (2007).
The one-electron autoxidation of human cytochrome P450 3A4.
  J Biol Chem, 282, 26865-26873.  
17686967 L.Wei, C.W.Locuson, and T.S.Tracy (2007).
Polymorphic variants of CYP2C9: mechanisms involved in reduced catalytic activity.
  Mol Pharmacol, 72, 1280-1288.  
16762915 I.G.Denisov, Y.V.Grinkova, B.J.Baas, and S.G.Sligar (2006).
The ferrous-dioxygen intermediate in human cytochrome P450 3A4. Substrate dependence of formation and decay kinetics.
  J Biol Chem, 281, 23313-23318.  
16407211 J.Sudhamsu, and B.R.Crane (2006).
Structure and reactivity of a thermostable prokaryotic nitric-oxide synthase that forms a long-lived oxy-heme complex.
  J Biol Chem, 281, 9623-9632.
PDB code: 2flq
15770070 H.Yasui, S.Hayashi, and H.Sakurai (2005).
Possible involvement of singlet oxygen species as multiple oxidants in p450 catalytic reactions.
  Drug Metab Pharmacokinet, 20, 1.  
16080215 P.Meinhold, M.W.Peters, M.M.Chen, K.Takahashi, and F.H.Arnold (2005).
Direct conversion of ethane to ethanol by engineered cytochrome P450 BM3.
  Chembiochem, 6, 1765-1768.  
15507439 T.W.Ost, and S.Daff (2005).
Thermodynamic and kinetic analysis of the nitrosyl, carbonyl, and dioxy heme complexes of neuronal nitric-oxide synthase. The roles of substrate and tetrahydrobiopterin in oxygen activation.
  J Biol Chem, 280, 965-973.  
15133020 D.J.Stuehr, J.Santolini, Z.Q.Wang, C.C.Wei, and S.Adak (2004).
Update on mechanism and catalytic regulation in the NO synthases.
  J Biol Chem, 279, 36167-36170.  
15560776 P.Hlavica (2004).
Models and mechanisms of O-O bond activation by cytochrome P450. A critical assessment of the potential role of multiple active intermediates in oxidative catalysis.
  Eur J Biochem, 271, 4335-4360.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.