Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen.

Rapid-scan and fixed-wavelength stopped-flow spectrophotometry were used to characterize the 5-methylphenazinium methyl sulfate (PMS)/reduced nicotinamide adenine dinucleotide couple at pH 7.4. Under anaerobic conditions, NADH reduces PMS to 5,10-dihydro-5-methylphenazine with a second order rate constant of 3.8 +/- 0.4 X 10(3) M-1 s-1. Oxygen reacts with 5,10-dihydro-5-methylphenazine to form PMS with a rate constant of about 180 M-1 s-1. When NADH reacts with PMS under aerobic conditions, a situation commonly encountered in carrying out routine enzymatic assays, the NADH reduction reaction and the O2 oxidation reaction proceed simultaneously with rate constants essentially the same as those determined for the two isolated reactions. The anaerobic photoreaction of PMS at pH = 7.4, which was studied by optical absorption spectroscopy, produces the 1-hydroxy-5-methylphenazinium cation (pyocyanine) and 5,10-dihydro-5-methylphenazine in nearly equal concentrations. When oxygen is present, the only detectable product is pyocyanine. These results, particularly the relatively slow rate of reaction between PMS and NADH, are used to point out potential complications in the use of the PMS/NADH couple.