title: Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr(103) affects rate and product distribution.
abstract: Mb (myoglobin) plus H2O2 catalyses the oxidation of various substrates via a peroxidase-like activity. A Y103F (Tyr103-->Phe) variant of human Mb has been constructed to assess the effect of exchanging an electron-rich oxidizable amino acid on the peroxidase activity of human Mb. Steady-state analyses of reaction mixtures containing Y103F Mb, purified linoleic acid and H2O2 revealed a lower total yield of lipid oxidation products than mixtures containing the wild-type protein, consistent with the reported decrease in the rate constant for reaction of Y103F Mb with H2O2 [Witting, Mauk and Lay (2002) Biochemistry 41, 11495-11503]. Irrespective of the Mb employed, lipid oxidation yielded 9(R/S)-HODE [9(R,S)-hydroxy-10E,12Z-octadecadienoic acid] in preference to 13(R/S)-HODE [13(R,S)-hydroxy-9Z,11E-octadecadienoic acid], while 9- and 13-keto-octadecadienoic acid were formed in trace amounts. However, lipid oxidation by the Y103F variant of Mb proceeded with a lower V(max) value and an increased K(m) value relative to the wild-type control. Consistent with the increased K(m), the product distribution from reactions with Y103F Mb showed decreased selectivity compared with the wild-type protein, as judged by the decreased yield of 9(S)-relative to 9(R)-HODE. Together, these data verify that Tyr103 plays a significant role in substrate binding and orientation in the haem pocket of human Mb. Also, the midpoint potential for the Fe(III)/(II) one-electron reduction was shifted slightly, but significantly, to a higher potential, confirming the importance of Tyr103 to the hydrogen-bonding network involving residues that line the haem crevice of human Mb.
authors.affiliation: Vascular Biology Group, ANZAC Research Institute, Hospital Road, Concord Repatriation General Hospital, Concord, NSW 2139, Australia.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Rayner
authors.forename: Roland
authors.initials: R
authors.lastname: Stocker
authors.forename: Peter A
authors.initials: PA
authors.lastname: Lay
authors.forename: Paul K
authors.initials: PK
authors.lastname: Witting
mesh_terms: D000818:Animals; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D019787:Linoleic Acid / Q000737:chemistry*; D015227:Lipid Peroxidation / Q000502:physiology; D009211:Myoglobin / Q000737:chemistry* / Q000502:physiology*; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry; D013237:Stereoisomerism; D014443:Tyrosine / Q000502:physiology*; D014907:Whales
chemical_list: D009211:Myoglobin; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D019787:Linoleic Acid; D006861:Hydrogen Peroxide