Study of the highly oxidizing centers in the metmyoglobin-H2O2 reaction

Metmyoglobin (MbFe III ) from horse heart forms an oxyferryl heme (Fe IV = O) and an unstable protein radical upon reaction with H 2 O 2 . Chemical modifications to the globin and heme have been reported as by-products of the reaction. Damage to the polypeptide was found to be linked to the Fe IV = O autoreduction process, with the extent of chemical modification increasing at lower pH. MbFe III was also found to demonstrate catalase-like activity after "priming" with 2 molar equivalents of H 2 O 2 . FTIR analysis of MbFe IV = O and MbFe III over the pH range 7 to 5.4 revealed minor changes in the amide I ' and II bands, indicating only small secondary structure changes. Thus, electron transfer pathways through the polypeptide to the heme are not expected to be perturbed by reaction with H 2 O 2 nor by pH changes. Covalent modification of His48 of Mb was performed with three ruthenium complexes (a 4 LRu; a = NH 3 ; L = NH 3 , pyridine, isonicotinamide). The reduction potentials (Ru III/II ) of the Mb-bound complexes were determined to be 77, 330, and 400 mV for the a 5 Ru, a 4 PyrRu, and a 4 IsnRu derivatives, respectively. The kinetics of Fe IV = O heme reduction in Mb by a 4 LRu II bound at the surface His48 were investigated using pulse radiolysis. The observed first-order rate constant (k obs1 ) decreased with increasing pH and reduction potential for the a 4 LRu centers. Rate-pD data obtained in D 2 O for the a 5 Ru derivative revealed the presence of an equilibrium isotope effect, and a pK a , of 5.7 (6.2 in D 2 O) was obtained for the acid-base group, which is assigned to the distal His64. A mechanism where protonation precedes ET provided a good fit of the kinetic data for the three a 4 LRu derivatives. Marcus theory analysis of the k ET (0.74, 1.8, 3.6 s -1 for L = Isn, Pyr, NH 3 ) extracted from the k obs1 's yielded a reorganization energy (n) of 1.8 eV for Ru II [arrow right] Fe IV = O ET in the a 4 LRu derivatives, but a n of 2.1 eV for the a 5 Ru derivative. From the latter, it is concluded that ET is strongly gated in the a 5 Ru derivative, and this is assumed to be the major reason for the low reactivity of Fe IV = O in Mb at high -x o . The location of protein-based radicals formed in the MbFe III /H 2 O 2 reaction was investigated by forming stable derivatives at the sites of radical formation. The spin adducts formed between Mb and the spin trap 2-methyl-2-nitrosopropan (MNP) were found to be stable upon reduction with ascorbate. Peptide mass mapping and sequencing by collisionally induced dissociation revealed that Tyr103 and Lys42 were modified in the MbFe III /H 2 O2/MNP reaction at neutral pH, and at pH 5 distal His64 was also modified