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Predicate | Object |
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
1-2
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pubmed:dateCreated |
1997-3-20
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pubmed:abstractText |
A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH-linked spectroscopic species. The electronic absorption and 1H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Soc. 114, 6592-6593; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1993) Biochemistry 32, 9807-9818]. Stopped-flow studies failed to detect even transient formation of a porphyrin-centered radical following addition of hydrogen peroxide to the Fe(III)-enzyme. The consequences of this drastic electrostatic modification of the active site on the steady-state kinetics of the variant are relatively minor.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Cytochrome-c Peroxidase,
http://linkedlifedata.com/resource/pubmed/chemical/Fungal Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Heme,
http://linkedlifedata.com/resource/pubmed/chemical/Hemeproteins,
http://linkedlifedata.com/resource/pubmed/chemical/Hydrogen Peroxide
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pubmed:status |
MEDLINE
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pubmed:month |
Jan
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pubmed:issn |
0014-2956
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
15
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pubmed:volume |
243
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
72-84
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pubmed:dateRevised |
2007-7-23
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pubmed:meshHeading |
pubmed-meshheading:9030724-Binding Sites,
pubmed-meshheading:9030724-Cytochrome-c Peroxidase,
pubmed-meshheading:9030724-Electron Spin Resonance Spectroscopy,
pubmed-meshheading:9030724-Fungal Proteins,
pubmed-meshheading:9030724-Heme,
pubmed-meshheading:9030724-Hemeproteins,
pubmed-meshheading:9030724-Hydrogen Peroxide,
pubmed-meshheading:9030724-Hydrogen-Ion Concentration,
pubmed-meshheading:9030724-Kinetics,
pubmed-meshheading:9030724-Magnetic Resonance Spectroscopy,
pubmed-meshheading:9030724-Oxidation-Reduction,
pubmed-meshheading:9030724-Protein Denaturation,
pubmed-meshheading:9030724-Saccharomyces cerevisiae,
pubmed-meshheading:9030724-Spectrum Analysis,
pubmed-meshheading:9030724-Temperature
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pubmed:year |
1997
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pubmed:articleTitle |
Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant.
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pubmed:affiliation |
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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