Reduction of methemoglobin by ferredoxin and ferredoxin-NADP reductase system.

The changes in absorption spectra between 450 and 650 nm during the reduction of methemoglobin A, (alpha 2+ beta 3+)2, and (alpha 3+ beta 2+)2 valency hybrids by the system including ferredoxin and ferredoxin-NADP reductase were studied under anaerobic conditions. During the reduction of methemoglobin A, the isosbestic points gradually shifted to different positions. These shifts were clearly observed in the presence of inositol hexaphosphate, i.e. the isosbestic points were initially observed at 525 and 603 nm, and these shifted to 529 and 599 nm, respectively, suggesting that the intermediate hemoglobins are produced during the process of the reaction. This was confirmed by the isoelectric focusing electrophoresis of the partially reduced methemoglobin solutions with ferredoxin-NADP reductase system on Ampholine-polyacrylamide gel plate. On the other hand, the absorption spectra of alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 changed with excellent isosbestic points during th reductive reaction by ferredoxin-NADP reductase system, i.e. (alpha 2+ beta 3+)2 at 526 and 601 nm and (alpha 3+ beta 2+)2 at 529 and 599 nm. From these results, the mechanism of methemoglobin reduction by ferredoxin-NADP reductase system was suggested. 1) There are two pathways for the reduction of methemoglobin including (formula, see text). 2) The beta chains of methemoglobin may be more susceptible to the reduction than the alpha chains in tetrameric methemoglobin, and thereby the (alpha 3+ beta 2+)2 valency hybrid accumulated at the halfway point of the reaction. 3) The shift in isosbestic points of methemoglobin reduction (525 nm leads to 529 nm, 603 nm leads to 599 nm) is due to the accumulation of (alpha 3+ beta 2+)2, whose isosbestic points during the reduction by the ferredoxin-NADP reductase systems were 529 and 599 nm.