Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr¹⁰³ affects rate and product distribution

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.