OPTICAL SPECTROPOTENTIOMETRIC RESOLUTION OF THE HEMES OF HYDROXYLAMINE OXIDOREDUCTASE - HEME QUANTITATION AND PH-DEPENDENCE OF E(M)

The hemes of hydroxylamine oxidoreductase (HAO) have been analyzed opt ically by potentiometric titrations using a low volume optically trans parent thin layer electrochemical cell. The electrochemical behavior o f the HAO monomeric unit has been interpreted by modeling the spectroe lectrochemical data at several wavelengths to eight one-electron Nerns t sites: seven c-type hemes and one P460 heme. Of the seven c-hemes, s ix show alpha-bands with absorption maxima at or near 553 nm. One c-he me has an alpha-band absorption maximum at 559 nm. The six c-553 hemes have midpoint potentials at pH 7.0 of +288, -10, -162, -192, -265 and -412 mV versus the normal hydrogen electrode (NHE). The c-559 heme ha s a midpoint potential (E(m)') at pH 7.0 of +11 mV versus NHE. The mid point potential of the P460 heme is at -260 mV versus NHE at pH 7.0. I n contrast, the midpoint potential for the P460 heme in another protei n, cytochrome P460, from the same organism is -402 mV versus NHE at pH 7.0. Midpoint potentials of the c-hemes show little, if any, pH depen dence over the range of pH 6-8. In contrast, E(m)' for the P460 heme c hanges with a slope of -60 mV/pH unit over the same range. Electrochem ical isolation of the P460 heme at pH 8.0 led to the discovery of a br oad spectroscopic feature centered near 740 nm that was assigned to th e oxidized P460 heme. Changes in the spectroelectrochemical behavior o f HAO after inactivation by H2O2 was almost exclusively restricted to the P460 heme of HAO. Both the 464-nm absorption band of the reduced P 460 heme and the 740-nm band of the oxidized heme were no longer prese nt. For the c-hemes, the only effect seems to be a slight shift in E(m )' for a single c-553 heme from -162 to -135 mV.