A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli. In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom. The Phe-221 → Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid. Significant differences between the mutant and wild-type enzymes can be detected spectroscopically. These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pKa of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms. The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by 1H-NMR data, and the loss of the πŐπ interaction between His-170 and Phe-221.
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January 2001
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Research Article|
January 08 2001
Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221→Met mutant
Barry D. HOWES;
Barry D. HOWES
*Dipartimento di Chimica, Università di Firenze, Via G. Capponi 9, 50121 Firenze, Italy,
‡School of Biological Sciences, University of Sussex, Brighton, BN1 9QG, U.K.
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Nigel C. VEITCH;
Nigel C. VEITCH
†Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, U.K.
‡School of Biological Sciences, University of Sussex, Brighton, BN1 9QG, U.K.
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Andrew T. SMITH;
Andrew T. SMITH
*Dipartimento di Chimica, Università di Firenze, Via G. Capponi 9, 50121 Firenze, Italy,
‡School of Biological Sciences, University of Sussex, Brighton, BN1 9QG, U.K.
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Christopher G. WHITE;
Christopher G. WHITE
*Dipartimento di Chimica, Università di Firenze, Via G. Capponi 9, 50121 Firenze, Italy,
‡School of Biological Sciences, University of Sussex, Brighton, BN1 9QG, U.K.
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Giulietta SMULEVICH
Giulietta SMULEVICH
1
*Dipartimento di Chimica, Università di Firenze, Via G. Capponi 9, 50121 Firenze, Italy,
‡School of Biological Sciences, University of Sussex, Brighton, BN1 9QG, U.K.
1To whom correspondence should be addressed (e-mail smulev@chim.unifi.it).
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Publisher: Portland Press Ltd
Received:
March 24 2000
Revision Received:
September 26 2000
Accepted:
October 30 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2001
2001
Biochem J (2001) 353 (2): 181–191.
Article history
Received:
March 24 2000
Revision Received:
September 26 2000
Accepted:
October 30 2000
Citation
Barry D. HOWES, Nigel C. VEITCH, Andrew T. SMITH, Christopher G. WHITE, Giulietta SMULEVICH; Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221→Met mutant. Biochem J 15 January 2001; 353 (2): 181–191. doi: https://doi.org/10.1042/bj3530181
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