| CO dissociation in cytochrome c peroxidase: site-directed mutagenesis shows that distal Arg 48 influences CO dissociation rates. | |
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MedLine Citation:
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PMID: 2176859 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast [CCP(MI)] and in several mutants in the heme binding pocket prepared by site-directed mutagenesis. The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly. Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form. Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here. In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI). Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly. In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms. Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS) |
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Authors:
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M A Miller; J M Mauro; G Smulevich; M Coletta; J Kraut; T G Traylor |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Biochemistry Volume: 29 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 1990 Oct |
Date Detail:
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Created Date: 1991-02-27 Completed Date: 1991-02-27 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 9978-88 Citation Subset: IM |
Affiliation:
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Department of Chemistry, University of California, San Diego, La Jolla 92093. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Arginine Binding Sites Carbon Monoxide / metabolism* Cytochrome-c Peroxidase / genetics, metabolism* Fungal Proteins / genetics, metabolism* Heme Hydrogen-Ion Concentration Kinetics Mutagenesis, Site-Directed Recombinant Proteins / metabolism Saccharomyces cerevisiae / enzymology, genetics |
| Grant Support | |
ID/Acronym/Agency:
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5 T32 AM07233-11/AM/NIADDK NIH HHS; GM10292-02/GM/NIGMS NIH HHS; HL13581/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Fungal Proteins; 0/Recombinant Proteins; 14875-96-8/Heme; 630-08-0/Carbon Monoxide; 74-79-3/Arginine; EC 1.11.1.5/Cytochrome-c Peroxidase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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