PDBsum entry 3ccx

Oxidoreductase (h2o2(a))

PDB id: 3ccx

Contents

Protein chain

291 a.a. *

Ligands

HEM

Waters ×102

* Residue conservation analysis

PDB id:

3ccx

Name:

Oxidoreductase (h2o2(a))

Title:

Altering substrate specificity at the heme edge of cytochromE C peroxidase

Structure:

CytochromE C peroxidase. Chain: a. Engineered: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932

Resolution:

2.30Å R-factor: 0.170

Authors:

S.K.Wilcox,G.M.Jensen,M.M.Fitzgerald,D.E.Mcree,D.B.Goodin

Key ref:

S.K.Wilcox et al. (1996). Altering substrate specificity at the heme edge of cytochrome c peroxidase. Biochemistry, 35, 4858-4866. PubMed id: 8664277 DOI: 10.1021/bi952929f

Date:

17-Mar-95 Release date: 10-Jul-95

Protein chain

P00431  (CCPR_YEAST) -  Cytochrome c peroxidase, mitochondrial from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 361 a.a.

Struc: 291 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.11.1.5 - cytochrome-c peroxidase.
• Reaction: 2 Fe(II)-[cytochrome c] + H2O2 + 2 H⁺ = 2 Fe(III)-[cytochrome c] + 2 H2O
• Cofactor: Heme

Heme (Bound ligand (Het Group name = HEM) matches with 95.45% similarity)

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

Added reference

DOI no: 10.1021/bi952929f

Biochemistry 35:4858-4866 (1996)

PubMed id: 8664277

Altering substrate specificity at the heme edge of cytochrome c peroxidase.

S.K.Wilcox, G.M.Jensen, M.M.Fitzgerald, D.E.McRee, D.B.Goodin.

ABSTRACT

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.