Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: Investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9-285 GHz) electron paramagnetic resonance spectroscopy

被引:28
作者
Fielding, Alistair J. [2 ]
Singh, Rahul [2 ]
Boscolo, Barbara [1 ]
Loewen, Peter C. [3 ]
Ghibaudi, Elena M. [1 ]
Ivancich, Anabella [2 ]
机构
[1] Univ Turin, Dipartimento Chim IFM, I-10125 Turin, Italy
[2] CEA, CNRS, URA 2096, IBITEC,Lab Hyperfrequences Metalloprot & Syst Spi, F-91191 Gif Sur Yvette, France
[3] Univ Manitoba, Dept Microbiol, Winnipeg, MB R3T 2N2, Canada
关键词
D O I
10.1021/bi801032k
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have combined the information obtained from rapid-scan electronic absorption spectrophotometry and multifrequency (9-295 GHz) electron paramagnetic resonance (EPR) spectroscopy to unequivocally determine the electronic nature of the intermediates in milk lactoperoxidase as a function of pH and to monitor their reactivity with organic substrates selected by their different accessibilities to the heme site. The aim was to address the question of the putative catalytic role of the protein-based radicals. This experimental approach allowed us to discriminate between the protein-based radical intermediates and [Fe(IV)=O] species, as well as to directly detect the oxidation products by EPR. The advantageous resolution of the g anisotropy of the Tyr(center dot) EPR spectrum at high fields showed that the tyrosine of the [Fe(IV)=O Tyr(center dot)] intermediate has an electropositive and pH-dependent microenvironment [g(x) value of 2.0077(0) at pH >= 8.0 and 2.0066(2) at 4.0 <= pH >= 7.5] possibly related to the radical stability and function. Two types of organic molecules (small aromatic vs bulkier substrates) allowed us to distinguish different mechanisms for substrate oxidation. [Fe(IV)=O Por(center dot+)] is the oxidizing species of benzohydroxamic acid, o-dianisidine, and o-anisidine via a heme-edge reaction and of mitoxantrone via a long-range electron transfer (favored at pH 8) not involving the tyrosyl radical, the formation of which competed with the substrate oxidation at pH 5. In contrast, the very efficient reaction with ABTS at pH 5 is consistent with [Fe(IV)=O Tyr(center dot)] being the oxidizing species. Accordingly, the identification of the ABTS binding site by X-ray crystallography may be a valuable tool in rational drug design.
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收藏
页码:9781 / 9792
页数:12
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