Photoreduction of methemoglobin by irradiation in the near-ultraviolet region

Ferric metHb can be photoreduced to the ferrous state by direct photoexcita tion in the near-ultraviolet region. In this research, we studied the mecha nism and facilitating conditions for the photoreduction and the resulting r estoration of O-2 binding. MetHb in phosphate-buffered saline or pure water in a CO atmosphere was photoreduced to form HbCO by illuminating the N ban d (365 nm), one of the porphyrin pi --> pi* transitions, whereas the photor eduction did not occur in Ar, N-2, or O-2. The transient absorption spectru m exhibited the generation of deoxyHb within 30 ns in both the CO and Ar at mospheres; however, only in CO did the subsequent CO binding inhibit the ba ck reaction. The photoreduction rate was dependent on the pH and ligand ani ons, showing that aquametHb in the high-spin state was predominant for the photoreduction. Axial ligand-to-metal charge-transfer (LMCT) bands overlap with the Soret and Q bands in metHb; however, the excitation of these bands showed little photoreduction, indicating that the contribution of these LM CT bands is minimal. Excitation of the N band significantly contributes to the photoreduction, and this is facilitated by the external addition of man nitol, hyaluronic acid, Trp, Tyr, etc. Especially, Trp allowed the photored uction even in an Ar atmosphere, and the reduced Hb can be converted to HbO (2) by O-2 bubbling. One mechanism of the metHb photoreduction that is prop osed on the basis of these results consists of a charge transfer from the p orphyrin ring to the central, ferric iron to form the porphyrin pi cation r adical and ferrous iron by the N band excitation, and the contribution of t he amino acid residues in the globin chain as an electron donor or an elect ron pathway.