Soluble cytochrome b 5 reductase from human erythrocytes.

I. An enzyme that catalyzes the reduction of erythrocyte cytochrome b 5 has been isolated from the supernatant fraction of erythrocyte hemolysates by chromatography on DEAE-cellulose, Amberlite CG-5 o, and Bio-Gel P-6o. 2. Erythrocyte cytochrome b 5 reductase has been shown to contain FAD. Incubation of the reductase in the absence of EDTA results in both the appearance of flavin fluorescence and the loss of enzymatic activity with time. 3. The reductase catalyzes the reduction of erythrocyte cytochrome b 5 5o times faster with NADH than with NADPH. The apparent Km of NADH was calculated to be 1.6. IO -~ M and the turnover number is 128o moles of cytochrome b 5 per min per mole of flavin. The reduction of electron acceptors proceeded in the following decreasing order of rate: K~Fe(CN)6, 2,6-dichlorophenolindophenol (DCIP), cytochrome b 5, methylene blue, cytochrome c, 03, oxidized glutathione, and methemoglobin. 4The FAD prosthetic group, the substrate specificity, and the effect of ionic strength, pH, and EDTA on activity suggest that the reductase is the same enzyme as NADH dehydrogenase I, the enzyme lacking in many cases of congenital methemoglobinemia. The properties of the reductase, including its molecular weight, are very similar to those of the cytochrome b 5 reductases solubilized from microsomes and mitochondria of other tissues.