A. Resonance Raman spectroscopy and optical spectroscopy were used to monitor the redox state of Heme A, having a and a3 centers, in cytochrome oxidase during potentiometric titrations. A novel approach was derived for obtaining simultaneous resonance Raman and optical absorption measurements of dilute protein solutions whose titrations curves are controlled potentiometrically. Quantitative Raman intensities, excited at 441.6 nm and measured as a function of solution voltage, yield the midpoint potential of the heme A centers. The techniques were first tested by deriving the well-known midpoint potential of cytochrome c. Cyanide- inhibited cytochrome oxidase was examined as a simplified model of the native enzyme in which the heme a3 is locked into the oxidized state. The spectroscopic analyses indicate that even when heme a3 cannot undergo oxidoreduction, the heme a centers show effective Em's near 260 and 350 mV. Measurements on native (unbound) cytochrome oxidase give two effective Em values near 260 and 350 mV for heme a and lower Em's near 260 and 350 mV for the heme a3 centers. These results argue against the Neoclassical model of cytochrome redox behavior in which the heme a and heme a3 centers are predicted to be equally reduced at all voltages. That is, these finding are consistent with recent reports of differential heme a and heme a3 redox behavior. B. A solid-state acousto-optic tunable filter (AOTF) was combined with krypton laser excitation, holographic filters and a photon-counting silicon avalanche photodiode detection to construct a miniaturized Raman spectrometer with no moving parts. The miniature AOTF spectrometer is used as an accessory to a Raman imaging microscope system in our laboratory. Although this spectrometer is optimized for the collection of Raman microspectra, it also functions as a microspectrometer for fluorescent studies.
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