PDBsum entry 2a1m

Oxidoreductase

PDB id: 2a1m

Contents

Protein chains

405 a.a. *

Ligands

HEM-OXY ×2

CAM ×2

TRS

Metals

__K ×3

Waters ×435

* Residue conservation analysis

PDB id:

2a1m

Name:

Oxidoreductase

Title:

Crystal structure of ferrous dioxygen complex of wild-type cytochrome p450cam

Structure:

Cytochrome p450-cam. Chain: a, b. Synonym: camphor 5-monooxygenase, p450cam. Engineered: yes. Mutation: yes

Source:

Pseudomonas putida. Organism_taxid: 303. Gene: camc. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.10Å R-factor: 0.191 R-free: 0.247

Authors:

S.Nagano,T.L.Poulos

Key ref:

S.Nagano and T.L.Poulos (2005). Crystallographic study on the dioxygen complex of wild-type and mutant cytochrome P450cam. Implications for the dioxygen activation mechanism. J Biol Chem, 280, 31659-31663. PubMed id: 15994329 DOI: 10.1074/jbc.M505261200

Date:

20-Jun-05 Release date: 05-Jul-05

Protein chains

P00183  (CPXA_PSEPU) -  Camphor 5-monooxygenase from Pseudomonas putida

Seq: 415 a.a.

Struc: 405 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.14.15.1 - camphor 5-monooxygenase.
• Reaction: 2 reduced [2Fe-2S]-[putidaredoxin] + (1R,4R)-camphor + O2 + 2 H⁺ = (1R,4R,5R)-5-hydroxycamphor + 2 oxidized [2Fe-2S]-[putidaredoxin] + H2O

2 × reduced [2Fe-2S]-[putidaredoxin] + (1R,4R)-camphor + O2 (Bound ligand (Het Group name = OXY) corresponds exactly) + 2 × H(+) = (1R,4R,5R)-5-hydroxycamphor + 2 × oxidized [2Fe-2S]-[putidaredoxin] + H2O

• Cofactor: Heme-thiolate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

DOI no: 10.1074/jbc.M505261200

J Biol Chem 280:31659-31663 (2005)

PubMed id: 15994329

Crystallographic study on the dioxygen complex of wild-type and mutant cytochrome P450cam. Implications for the dioxygen activation mechanism.

S.Nagano, T.L.Poulos.

ABSTRACT

Two key amino acids, Thr252 and Asp251, are known to be important for dioxygen activation by cytochrome P450cam. We have solved crystal structures of a critical intermediate, the ferrous dioxygen complex (Fe(II)-O2), of the wild-type P450cam and its mutants, D251N and T252A. The wild-type dioxygen complex structure is very much the same as reported previously (Schlichting, I., Berendzen, J., Chu, K., Stock, A. M., Maves, S. A., Benson, D. E., Sweet, R. M., Ringe, D., Petsko, G. A., and Sligar, S. G. (2000) Science 287, 1615-1622) with the exception of higher occupancy and a more ordered structure of the iron-linked dioxygen and two "catalytic" water molecules that form part of a proton relay system to the iron-linked dioxygen. Due to of the altered conformation of the I helix groove these two waters are missing in the D251N dioxygen complex which explains its lower catalytic activity and slower proton transfer to the dioxygen ligand. Similarly, the T252A mutation was expected to disrupt the active site solvent structure leading to hydrogen peroxide formation rather than substrate hydroxylation. Unexpectedly, however, the two "catalytic" waters are retained in the T252A mutant. Based on these findings, we propose that the Thr(252) accepts a hydrogen bond from the hydroperoxy (Fe(III)-OOH) intermediate that promotes the second protonation on the distal oxygen atom, leading to O-O bond cleavage and compound I formation.

Selected figure(s)

Figure 1.
FIG. 1. A, the structure of P450cam highlighting the I helix and the catalytically important residues, Thr252 and Asp251. B, the P450 oxygen activation mechanism. Electron and proton transfer to the ferrous dioxygen complex gives the Fe(III)-OOH hydroperoxy intermediate. A second protonation of the distal oxygen atom leads to heterolysis of the dioxygen O-O bond and formation of Fe(IV)=O, the active hydroxylating species.

Figure 4.
FIG. 4. Possible hydrogen bond network for the P450cam hydroperoxy intermediate. The distances between heteroatoms are taken from molecule B of our WT dioxygen complex structure.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 31659-31663) copyright 2005.

Figures were selected by an automated process.