Resonance Raman spectroscopy of cytochrome oxidase using Soret excitation: selective enhancement, indicator bands, and structural significance for cytochromes a and a3

Resonance Raman studies of oxidized and reduced cytochrome oxidase and liganded derivatives of the oxidized enzyme have been performed by using direct-Soret excitation at 413.1 and 406.7 nm, as well as near-Soret excitation (457.9 nm) and alpha-band excitation (604.6 nm). The Soret results clearly show selective enhancement of Raman modes of the hemes of cytochromes a and a3, depending upon the excitation wavelength chosen. For the preparations employed in this study, photoreduction of cytochrome oxidase in the laser beam was not a significant problem. Resonance Raman frequencies sensitive to oxidation state and spin state or core expansion of the a and a3 hemes are identified and correlated with those previously identified for other heme proteins. An unusual low-frequency (less than 500 cm(-1)) spectrum is observed for oxidized high-spin cytochrome a3, which may be due to axial nonheme structures in this cytochrome.     

Core protein I as an artifact in complex III.

SDS electrophoresis gels of complex III from yeast mitochondria were run after incubation of the enzyme at several different temperatures. It was found that the intensity of the more slowly moving core protein band was substantially affected by the incubation temperature. Four low molecular weight polypeptides were eluted from gels electrophoresed after preincubation of the enzyme at 15° C for 12 hours. These polypeptides were then incubated for 5 minutes at 100° followed by SDS gel electrophoresis. A polypeptide with the same molecular weight as the anomalous core protein was resolved.     

Hydrolysis of triphosphoinositides by a soluble fraction of Crithidia fasciculata.

Homogenates of Crithidia fasciculata (a species of Trypanosomidae) were shown to contain a phosphatase (EC 3.1.3.36) and a phosphodiesterase (EC 3.1.4.11) which hydrolyse triphosphoinositides. Approximately 30% of the diesterase and most of the phosphatase are present in the soluble fraction. The triphosphoinositide phosphatase is specifically dependent upon Mg(2+) and is stable to storage with or without freezing. The triphosphoinositide phosphodiesterase requires Ca(2+) and is inactivated during storage. Both activities are maximal in the presence of cetyltrimethylammonium bromide and require protection or reactivation by GSH or dithiothreitol. Unlike similar mammalian enzymes the protozoal triphosphoinositide phosphatase does not hydrolyse diphosphoinositides. The two enzymes may be separated by (NH4)2SO4 fractionation and gel filtration on Sephadex G-200.