Reduction of methemoglobin via electron transfer from photoreduced flavin: Restoration of O₂-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH₂ by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O₂ binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Φ) of 0.17, that is higher than the method of direct photoexcitation of heme (Φ = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O₂ binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 μ (close to capillary diameter in tissue, 5 μ), and visible light irradiation (221 mW/cm²) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH₂ reacted with O₂ to produce H₂O₂, which was detected by the fluorescence measurement of the reaction of H₂O₂ and p-nitrophenylacetic acid. However, the amount of H₂O₂ generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH₂ was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.