METAL-CATALYZED OXIDATION OF HISTIDINE IN HUMAN GROWTH-HORMONE - MECHANISM, ISOTOPE EFFECTS, AND INHIBITION BY A MILD DENATURING ALCOHOL

Metal-catalyzed oxidation of proteins represents an important pathway of post-translational modification. We utilized human growth hormone ( hGH), a protein with a well defined metal-binding site, to study the d etailed mechanism of metal-catalyzed oxidation by ascorbate/Cu(II)/O-2 . Particularly His(18) and His(21) within the metal-binding site were oxidized, predominantly to S-oxo-His with the incorporated oxygen orig inating from molecular oxygen, based on amino acid analysis, tryptic m apping, mass spectrometry, isotopic labeling, and H-1 NMR. The anaerob ic reduction of a hGH/Cu(II) mixture by ascorbate generated a hGH-Cu(I ) complex with NMR spectral features different from those of native hG H and hGH/Cu(II). The anaerobic reaction of this hGH-Cu(I) complex wit h hydrogen peroxide resulted in the oxidation of His(18) and His(21), suggesting that a fraction of Cu(I) was bound at the metal-binding sit e of hGH. Site-specific oxidation of hGH required an intact metal-bind ing site and could largely (about 80%) be inhibited by the presence of greater than or equal to 28% (v/v) 1-propanol which appears (i) to pe rturb the metal-binding site and (ii) to interact with a reactive oxyg en species formed at the perturbed metal-binding site. The inhibition by 1-propanol-d(7) (CD3CD2CD2OH) was significantly lower than that by 1-propanol-h(7) with [residual hGH](1-propanol-h7)/[residual hGH](1-pr opanol-d7) = 1.95 at 30% (v/v) 1-propanol, reflecting a kinetic isotop e effect close to that for the reaction of a hydroxyl radical with C-a lpha-HID bonds of methanol, suggesting the involvement of a hydroxyl r adical-like species in the oxidation of His.