EPR investigation of compound I in Proteus mirabilis and bovine liver catalases: Formation of porphyrin and tyrosyl radical intermediates

被引:133
作者
Ivancich, A
Jouve, HM
Sartor, B
Gaillard, J
机构
[1] CEA GRENOBLE,SCPM,SCIB,DEPT RECH FONDAMENTALE MAT CONDENSEE,F-38054 GRENOBLE,FRANCE
[2] CEA GRENOBLE,DEPT BIOL MOL & STRUCT,BMC,F-38054 GRENOBLE,FRANCE
[3] CEA GRENOBLE,CNRS,LAB ENZYMOL MOL,INST BIOL STRUCT JEAN PIERRE EBEL,F-38027 GRENOBLE,FRANCE
关键词
D O I
10.1021/bi970886s
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
Compound I of Proteus mirabilis and bovine liver catalases (PMC and BLC, respectively) were studied combining EPR spectroscopy and the rapid-mix freeze-quench techniques. Both enzymes, when treated with peroxyacetic acid, form a catalytic intermediate which consists of an oxoferryl ?yl porphyrin pi-cation radical. In PMC this intermediate is semistable, and an unexpected reversible equilibrium under pH influence takes place between two forms of compound I with different coupling between the oxoferryl and the porphyrin pi-cation radical. At acid pH, one form has a ferromagnetic character as in Micrococcus luteus compound I. At neutral pH, another form with a much smaller coupling, reminiscent of the horse radish peroxidase compound I, is detected. The approximate midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pK(a) value of an histidyl residue. The residues possibly involved in the transformation are discussed in terms of the known structure of PMC compound I. The EPR spectrum of BLC compound I (pH 5.6), obtained in the millisecond time scale (40 ms), also showed a Mixture of two forms which, most probably, correspond to two different magnetic exchange interactions, as in the case of PMC. Taken together, the low-temperature electronic absorption and the EPR spectra of BLC compound I formed in the 0.04-15 s range show that the porphyrin pi-cation radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to the C-2,C-6 and C-3,C-5 ring protons and the beta-methylene protons of the purported tyrosyl radical. Candidates for such a tyrosyl radical are discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor,
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页码:9356 / 9364
页数:9
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