Intramolecular Translocation of the Protein Radical Formed in the. Reaction of Recombinant Sperm Whale Myoglobin with H202*. (Received for publication ...
Vol. 267, No. 13,Issue of May 5, pp. 8827-8833,1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Intramolecular Translocationof the ProteinRadical Formed inthe Reaction of Recombinant Sperm Whale Myoglobin with H202* (Received for publication, December 10, 1991)
Angela Wilksand PaulR. Ortiz deMontellano$ From the Department of Pharmaceutical Chemistry. School of Pharmacy, and Liver Center, Universityof California, S a n Francisco, California94143-0446
A sperm whale myoglobin gene containing multiple (6), ribonucleotide reductase (7, a), pyruvate lyase (9), galacunique restriction sites has been constructed in pUC tose oxidase (lo), and prostaglandin H synthase (11).A ty18 by sequential assembly of chemically synthesized rosine-centered radical is specificallyinvolvedinsome of oligonucleotide fragments. Expression of the gene in these enzymatic reactions (7, 8, 10, 11). Escherichia coli DH6a cells yields protein that is idenMyoglobin is not ordinarily a catalytic protein but does tical to native sperm whale myoglobin except that it support the HzOz-dependent oxidation of many substrates retains the terminal methionine. Site-specific muta- (12), including olefin epoxidation, a cytochrome P450-like genesis has been used to prepareall the possible tyro- reaction (13). Catalysis of these reactions is associated with sine phenylalanine mutantsof the recombinant myo- the formation of a ferryl (Fe’” = 0) species (14, 15). This globin, including the three single mutants at Tyr-103, ferryl species retains one of the twooxidizing equivalents - 146, and-161, thethree double mutants, and the tripleprovided by reaction with HzOz, but the second oxidizing mutant. All of the mutant proteins are stable except equivalent is dissipated bypoorly understood mechanisms the Tyr-103mutant. Introductionof a second mutation (Lys-102 4 Gln) stabilizes the Tyr-103 mutant. Ab- (14,15). EPR studies indicate that a protein radical is formed sorption spectroscopy suggests that the active sites of in the reactions of H202with sperm whale metmyoglobin (16, 17), horse metmyoglobin (14, la), horse methemoglobin (17), the mutant proteins are intact. EPR and absorption human methemoglobin (19, 20), and bovine methemoglobin spectroscopy show that all theproteins, including the triple mutant devoid of tyrosine residues, react with (21). Unlike the equimolar protein radical and ferryl complex HzOz to give a ferryl species and a protein radical. The produced in the reaction of cytochrome c peroxidase with presence of a protein radical in all the mutants suggests H202(6), the detectable radical signal in myoglobin accounts that theradical center is readilytransferred from one for no morethan 50% of the second oxidation equivalent (15). amino acid to another. Cross-linking studies show, EPRdata suggest that the protein radical resides on an however, that protein dimers are only formed when aromatic residue, most likely a tyrosine, although some eviTyr-161 is present. Tyr-103, shown earlier to be the dence exists that theradical may first reside transiently on a residue that primarily cross-links to Tyr-161 (Tew, histidine or other aromatic residue (17, 22). There are three D., and Ortiz de Montellano, P. R. (1988) J. Biol. tyrosine residues in sperm whale myoglobin (Tyr-103, TyrChem. 263, 17880-17886), is not essential for cross- 146,Tyr-1511,two in horse myoglobin (Tyr-103, Tyr-146), linking. Electrqn transfer from Tyr-151 to the heme, and only one in red kangaroo myoglobin (Tyr-146) (23). which are 12 A apart, occurs in the absence of the Reaction with H202results in covalent dimerization of sperm intervening tyrosines at positions 103 and 146. The present studiesshow that theperoxide-generated myo- whale (24) but nothorse (24) or red kangaroo (25) myoglobins. globin radical readily exchanges between remote loci, Analysis of the sperm whale reaction by tryptic digestion and including non-tyrosine residues, butprotein cross- peptide sequencing showed that dimerization primarily inlinking only occurs when radical density is located on volves cross-linking of Tyr-103 of one myoglobin chain with Tyr-151 of the second (25). The heme group of HzOz-treated Tyr-161. horse myoglobin is also covalently bound to the protein through Tyr-103 (26). Further evidence for dissipation of the second oxidizing equivalent through protein radical mechaThe biochemistry of protein radicals has become of increas- nisms comes from parallel work showing that a substantial ing interest as their pathological roles in lipid peroxidation proportion of the oxygen incorporated into the epoxide in the (1, 2) and in the structural perturbations of proteins that HzO2-dependent epoxidation of simple olefins comesfrom cause their proteolytic degradation (3,4) have become appar- molecular oxygen (13). The proposed mechanism for these ent. The interest in protein radicals also stems from their epoxidations involves co-oxidation by the peroxy radical genproposed roles in the normal catalytic mechanisms of a grow- erated when oxygen adds to theprotein radical, a mechanism ing number of enzymes (5), including cytochrome c peroxidase supported by the fact that 4-methylphenol, a stand-in for tyrosine, mediates the epoxidation of styrene by horseradish * This work was supported by Grant DK30297 from the National peroxidase and H,Oz (27). Institutes of Health. Instrumentsand facilities used in this work were Sperm whale myoglobin is ideal for the analysis of strucsupported in part by National Institutes of Health Grant GM32488 ture-function relationships pertinent to the biochemistry of and the University of California, San Francisco Liver Center Grant protein radicals because it is a small, rugged protein and 5 P30 DK26743.The costs of publication of this article were defrayed highly refined x-ray crystal structures are available for the in part by the payment of page charges. This article must therefore native (28) and recombinant proteins (29). We report here be hereby marked “aduertisement” in accordance with 18 U.S.C. chemical synthesis of a gene encoding sperm whale myoglobin, Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. its expression in Escherichia coli, successful construction and
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expression of all the possible mutants in which the tyrosine residues are replaced by phenylalanine, and examination of the EPR properties and susceptibility to dimerization of the expressed proteins.
(pH 6.0). The fractions containing the myoglobin (as judged by the absorbance at 408 nm) were pooled and concentrated. The protein was further purified by FPLC on a Mono S HR-10 column using a linear gradient of 50 mM sodium phosphate (pH 6) to 75 mM sodium phosphate (pH 8) over 45 min. The protein was judged pure as monitored by SDS-PAGE. The purified myoglobin (10 pg) was subjected to automated Edman degradation on an Applied Biosystems MATERIALS ANDMETHODS model 470A protein sequenator at the University of California, San General Methods-Plasmid purification, subcloning, and bacterial Francisco, Biomolecular Resource Center. transformation were carried out as described (30). Deionized, double Construction of the Mutants-The mutants were constructed by glass-distilled, Chelex-treated water was used for all biochemical removing the section of the gene containing the codon to be changed. experiments. Chelexwas from Bio-Rad. Oligonucleotides (80-108 The 40-bp XbaIISacI fragment in pMb221 was replaced with a base pairs) were synthesized at the University of California, San synthetic DNA cassette coding the change from Tyr-146 to Phe-146. Francisco, Biomolecular Resource Center using an Applied Biosys- The procedure was repeated for the 40-bp SacIIHindIII fragment for tems 380BDNA synthesizer. Oligonucleotides were purified on Tyr-151 to Phe-151. The Tyr-103 to Phe-103 and the double mutant PAGE,' 12% acrylamide, 8 M urea gels (31). DNA was eluted from Lys-102 to Gln-102/Tyr-103 to Phe-103 were constructed by replacing the gels in 0.5 M ammonium acetate and desalted by passage through the XhoIISphI fragment with a 34-base pair piece containing the a Pharmacia LKB Biotechnology Inc. NAP-25 column with water as respective codon changes. The double tyrosine mutants Y103/146F, the eluant. Oligonucleotides
