Abstract
Reaction of sperm whale metmyoglobin (SwMb) with H2O2 produces a ferryl (MbFeIV=O) species and a protein radical and leads to the formation of oligomeric products. The ferryl species is maximally formed with one equivalent of H2O2, and the maximum yields of the dimer (28%) and trimer (17%) with 1 or 2 eq. Co-incubation of the SwMb Y151F mutant with native apoSwMb and H2O2 produced dimeric products, which requires radical transfer from the nondimerizing Y151F mutant to apoSwMb. Autoreduction of ferryl SwMb to the ferric state is biphasic with t = 3.4 and 25.9 min. An intramolecular autoreduction process is implicated at low protein concentrations, but oligomerization decreases the lifetime of the ferryl species at high protein concentrations. A fraction of the protein remained monomeric. This dimerization-resistant protein was in the ferryl state, but after autoreduction it underwent normal dimerization with H2O2. Proteolytic digestion established the presence of both dityrosine and isodityrosine cross-links in the oligomeric proteins, with the isodityrosine links primarily forged by Tyr151-Tyr151 coupling. The tyrosine content decreased by 47% in the dimer and 14% in the recovered monomer, but the yields of isodityrosine and dityrosine in the dimer were only 15.2 and 6.8% of the original tyrosine content. Approximately 23% of the lost tyrosines therefore have an alternative but unknown fate. The results clearly demonstrate the concurrence of intra- and intermolecular electron transfer processes involving Mb protein radicals. Intermolecular electron transfers that generate protein radicals on bystander proteins are likely to propagate the cellular damage initiated by the reaction of metalloproteins with H2O2.
Highlights
Protein radicals, once generated, can participate in catalytic processes or can lead to cross-linking of amino acid residues, covalent bonding of prosthetic groups, formation of protein peroxides, and cleavage of the protein backbone (21)
It is not known whether the protein radicals formed in the LPO catalytic cycle have a biological function other than their probable involvement in autocatalytic covalent attachment of the prosthetic heme group to the protein (33), but we recently provided evidence that the LPO radical can be transferred to metMb (34)
We have examined in greater detail the protein radicals formed in the reaction of sperm whale metmyoglobin (SwMb) with H2O2 in order to quantitatively evaluate their fate and to determine the extent to which the protein radical undergoes intra- and intermolecular transfer from one site to another
Summary
Once generated, can participate in catalytic processes or can lead to cross-linking of amino acid residues, covalent bonding of prosthetic groups, formation of protein peroxides, and cleavage of the protein backbone (21). As proposed for Mb, this Compound I intermediate decays to a second “Compound I” species that retains the FeIVϭO but has a protein rather than porphyrin radical (30, 31) Formation of this second Compound I structure results in dimerization of LPO via a dityrosine cross-link involving Tyr289 (32). Unpaired electron density is shown by spin trapping and EPR studies to reside at sites in addition to Tyr289 (32) It is not known whether the protein radicals formed in the LPO catalytic cycle have a biological function other than their probable involvement in autocatalytic covalent attachment of the prosthetic heme group to the protein (33), but we recently provided evidence that the LPO radical can be transferred to metMb (34)
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