title: Engineering tyrosine-based electron flow pathways in proteins: the case of aplysia myoglobin.
abstract: Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate.  Aplysia fasciata  myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed.
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom. reedb@essex.ac.uk
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Reeder
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Svistunenko
authors.forename: Chris E
authors.initials: CE
authors.lastname: Cooper
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001048:Aplysia / Q000235:genetics* / Q000378:metabolism; D050356:Lipid Metabolism; D008958:Models, Molecular; D009154:Mutation; D009211:Myoglobin / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D014443:Tyrosine / Q000235:genetics* / Q000378:metabolism
chemical_list: D009211:Myoglobin; D014443:Tyrosine; D010649:Phenylalanine