: The objectives of the project were to compare oxidative metabolism of marine organisms with that in the well studied systems such as mammalian heart tissue, to map and explore the bioenergetic systems of representative marine organisms, and to 'cross reconstitute' the respiratory chain by components from marine organisms with those from mammalian heart. Almost essential for the above studies was the search for new methodology for studying bioenergetics.

It is now generally agreed that mitochondrial succinate dehydrogenase (SIE) (1) consists of two subunits ~icn are separable by SDS-PllGE. 'Ihe larger subunit oontains oovalently linked FAD with 1ID1ecular weight of about 70,000 knom as Fp and the smaller cne approximately 27,000 called Ip oontains cnly an iron-sulfur cluster without flavin. Ql the other hand, the distribution of iroo-sulfur clusters is sateWhat still not canpletely settled. Previously, it was tho\JJht that Fp oontains FAD together wi th two 2Fe2S centers and that Ip cxmtains ooe 4Fe4S center (4). Very recently, fran J;ilysical xrethods, a claim has been made that Fp consists of a 2Fe2S center and a 4Fe4S cluster, ~ereas Ip oonsists of a 3FeXS cluster (2, 3).

This chapter describes the preparation of hinge protein and its requirement for interaction of cytochrome c with cytochrome c1. The hinge protein is prepared by sequential resolution of the respiratory chain. Succinate-cytochrome-c reductase is isolated from the submitochondrial particles from where two-band cytochrome c1 is obtained. The hinge protein is prepared from two-band cl. The hinge protein is insensitive to temperature, urea, and low pH. However, when the preparation is kept in concentrated form for a few days, it tends to become turbid and evidently forms self-associated polymers. The hinge protein is quite different from the standard nonmembranous proteins employed for calibration of the gel filtration column for molecular weight. The principle of the determination of the hinge protein is based on the formation of the cytochrome c1-c-hinge protein complex in 1:1:1 ratio. The increase of molecular ellipticity is linear to the amount of the hinge protein in the system.

This book contains the proceedings of a symposium on oxidases and related redoxsystems. Topics covered include: Oxidases and related redoxsystems, Flavoprotein oxidases and oxygenases, Peroxidases, and Cytochrome P-450 and related proteins.

This chapter describes the Keilin–Hartree's heart muscle preparation. The heart muscle preparation has been used in the study of intracellular respiration and as a starting material for large-scale isolations of respiratory components. It is a particulate suspension of physically disintegrated mitochondrial membrane. The manipulations for the preparation are simple and the yield is high. A number of criteria may be used for the determination of the activity. Among them, oxidation of succinate or NADH by molecular oxygen is useful and informational. The following describe a manometric method for succinate oxidase and a polarographic procedure for NADH oxidase. Keilin originally employed the method of the preparation for his studies of intracellular respiration. Later Keilin and Hartree standardized the preparation. Subsequently, because of the availability of high-speed centrifuges and adaptations for special purposes, some variances in manipulations have been introduced. Four methods of preparation are described in this chapter.

This chapter discusses the hydrogen and electrons successively transfer through a number of oxidation–reduction carriers ultimately to oxygen. Energy derived from the reaction is utilized by the organism through energy coupling and transduction. The chapter discusses the mitochondrial hydrogen and electron transfer that occur in the inner membrane. The sequence of hydrogen and electron transfer can be deduced from the results of thermodynamic and kinetic studies. The former alone cannot determine the path; both concentrations of the components and the so-called energy barrier (activation energy) must also be considered. Results from kinetics are perhaps most direct and reliable. The reliability of the results is, needless to say, dependent upon the accurate selection of wave lengths used without interference by other substances present. The determinations of relatively rapid reactions possess some practical difficulties, even with special instruments.

A highly purified cytochrome b−c1 complex which is free of QPs (see BBRC 78, 259 and 79, 939) yields 20–30% of semiubiquinone (based on the total Q content in the system) in the presence of catalytic amounts of QPs and succinate dehydrogenase (at or lower than 2 × 10−9 M. The radical shows typical g = 2.00 signals with line widths of 8 and 9 gauss, respectively, at about 22°C and 77°K. The appearance of the radicals approximately parallels that of b reduction but not its disappearance. However, addition of theonytrifluoroacetone or antimycin A immediately abolishes both radical formation and b reduction. These and other observations indicate that the true carrier property of Q is through its binding with proper proteins but not the protei-free form.

This chapter provides information on electron paramagnetic resonance (EPR) and other properties of succinate dehydrogenase. EPR characteristics of iron–sulfur centers in reconstitutively active and inactive succinate dehydrogenase preparations are also summarized in the chapter. Three distinct iron–sulfur centers (clusters) and flavin free radicals are identified in the succinate dehydrogenase using EPR spectroscopy under various conditions. Two iron–sulfur centers show EPR signals in the reduced state, similar to plant or bacterial ferredoxins (Fd); the third center is paramagnetic in the oxidized state, as in case of Chromatium high-potential iron–sulfur protein (HiPIP). EPR signals of the HiPIP-type center (designated center S-3) are readily detectable in particulate succinate-Q reductase. The temperature affects the spectra of iron–sulfur centers in three major ways by decreasing the linewidth. At low temperatures, however, the signal may saturate at low power levels, decreasing the maximum intensity obtainable. Therefore it is necessary to examine power dependence of the signal at different temperatures.