2X08

cytochrome c peroxidase: ascorbate bound to the engineered ascorbate binding site

Summary for 2X08

• Entry DOI: 10.2210/pdb2x08/pdb
• Related: 1A2F 1A2G 1AA4 1AC4 1AC8 1AEB 1AED 1AEE 1AEF 1AEG 1AEH 1AEJ 1AEK 1AEM 1AEN 1AEO 1AEQ 1AES 1AET 1AEU 1AEV 1BEJ 1BEK 1BEM 1BEP 1BEQ 1BES 1BJ9 1BVA 1CCA 1CCB 1CCC 1CCE 1CCG 1CCI 1CCJ 1CCK 1CCL 1CCP 1CMP 1CMQ 1CMT 1CMU 1CPD 1CPE 1CPF 1CPG 1CYF 1DCC 1DJ1 1DJ5 1DS4 1DSE 1DSG 1DSO 1DSP 1EBE 1JCI 1JDR 1KOK 1KRJ 1KXM 1KXN 1MK8 1MKQ 1MKR 1ML2 1RYC 1S6V 1SDQ 1SOG 1STQ 1U74 1U75 1Z53 1ZBY 1ZBZ 2B0Z 2B10 2B11 2B12 2BCN 2CCP 2CEP 2CYP 2GB8 2PCB 2PCC 2V23 2V2E 2X07 2XIL 2XJ5 2XJ8 3CCP 3CCX 4CCP 4CCX 5CCP 6CCP 7CCP
• Descriptor: CYTOCHROME C PEROXIDASE, MITOCHONDRIAL, ASCORBIC ACID, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• Functional Keywords: oxidoreductase, metal-binding
• Biological source: SACCHAROMYCES CEREVISIAE (BAKER'S YEAST)
• Total number of polymer chains: 1
• Total formula weight: 34202.72

Authors

Murphy, E.J.,Metcalfe, C.L.,Gumiero, A.,Raven, E.L.,Moody, P.C.E. (deposition date: 2009-12-07, release date: 2010-11-10, Last modification date: 2023-12-20)

Primary citation

Murphy, E.J.,Metcalfe, C.L.,Basran, J.,Moody, P.C.,Raven, E.L.
Engineering the substrate specificity and reactivity of a heme protein: creation of an ascorbate binding site in cytochrome c peroxidase.
Biochemistry, 47:13933-13941, 2008

PubMed Abstract: The binding of substrates to heme enzymes has been widely assumed to occur at the so-called delta-heme edge. Recently, however, a number of examples have appeared in which substrate binding at an alternative site, the gamma-heme edge, is also possible. In previous work [Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307], we showed that binding of ascorbate to ascorbate peroxidase occurred at the gamma-heme edge. Here, we show that the closely related cytochrome c peroxidase enzyme can duplicate the substrate binding properties of ascorbate peroxidase through the introduction of relatively modest structural changes at Tyr36 and Asn184. Hence, crystallographic data for the Y36A/N184R/W191F triple variant of cytochrome c peroxidase shows ascorbate bound to the gamma-heme edge, with hydrogen bonds to the heme propionate and Arg184. In parallel mechanistic studies in variants incorporating the W191F mutation, we show that a transient porphyrin pi-cation radical in Compound I of cytochrome c peroxidase, analogous to that observed in ascorbate peroxidase, is competent for ascorbate oxidation but that under steady state conditions this intermediate decays too rapidly to sustain efficient turnover of ascorbate. The results are discussed in terms of our more general understanding of substrate oxidation across other heme proteins, and the emerging role of the heme propionates at the gamma-heme edge.

PubMed: 19061385
DOI: 10.1021/bi801480r

Experimental method

X-RAY DIFFRACTION (2.01 Å)