EPR INVESTIGATION OF COMPOUND-I IN PROTEUS-MIRABILIS AND BOVINE LIVERCATALASES - FORMATION OF PORPHYRIN AND TYROSYL RADICAL INTERMEDIATES

Compound I of Proteus mirabilis and bovine liver catalases (PMC and BL C, respectively) were studied combining EPR spectroscopy and the rapid -mix freeze-quench techniques. Both enzymes, when treated with peroxya cetic acid, form a catalytic intermediate which consists of an oxoferr yl ?yl porphyrin pi-cation radical. In PMC this intermediate is semist able, and an unexpected reversible equilibrium under pH influence take s place between two forms of compound I with different coupling betwee n the oxoferryl and the porphyrin pi-cation radical. At acid pH, one f orm 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 approxima te midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pK(a) value of an histidyl residue. The residues possib ly 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 Mixt ure of two forms which, most probably, correspond to two different mag netic exchange interactions, as in the case of PMC. Taken together, th e low-temperature electronic absorption and the EPR spectra of BLC com pound I formed in the 0.04-15 s range show that the porphyrin pi-catio n radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to th e C-2,C-6 and C-3,C-5 ring protons and the beta-methylene protons of t he purported tyrosyl radical. Candidates for such a tyrosyl radical ar e discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor,