The mechanism whereby cytochrome £ oxidase catalyses elec-. tron transfer from cytochrome £ to oxygen remains an unsolved problem. Polarographic and spectrophotometric activity measurements of purified, particulate and soluble forms of beef heart mitochondrial cytochrome c oxidase presented in this thesis confirm the following characteristics of the steady-state kinetics with respect to cytochrome £: (1) oxidation of ferrocytochrome c is first order under all conditions. -(2) The relationship between sustrate concentration and velocity is of the Michaelis-Menten type over a limited range of substrate. concentrations at high ionic strength. (3) ~he reaction rate is independent from oxygen concentration until very low levels of oxygen. (4) "Biphasic" kinetic plots of enzyme activity as a function of substrate concentration are found when the range of cytochrome c concentrations is extended; the biphasicity ~ is more apparent in low ionic strength buffer. These results imply two binding sites for cytochrome £ on the oxidase; one of high affinity and one of low affinity with Km values of 1.0 pM and 3.0 pM, respectively, under low ionic strength conditions. (5) Inhibition of the enzymic rate by azide is non-c~mpetitive with respect to cytochrome £ under all conditions indicating an internal electron transfer step, and not binding or dissociation of £ from the enzyme is rate limiting. The "tight" binding of cytochrome '£ to cytochrome c oxidase is confirmed in column chromatographic experiments. The complex has a cytochrome £:oxidase ratio of 1.0 and is dissociated in media of high ionic strength. Stopped-flow spectrophotometric studies of the reduction of equimolar mixtures and complexes of cytochrome c and the oxidase were initiated in an attempt to assess the functional relevance of such a complex. Two alternative routes -for reduction of the oxidase, under conditions where the predominant species is the £ - aa3 complex, are postulated; (i) electron transfer via tightly bound cytochrome £, (ii) electron transfer via a small population of free cytochrome c interacting at the "loose" binding site implied from kinetic studies. It is impossible to conclude, based on the results obtained, which path is responsible for the reduction of cytochrome a. The rate of reduction by various reductants of free cytochrome £ in high and low ionic strength and of cytochrome £ electrostatically bound to cytochrome oxidase was investigated. Ascorbate, a negatively charged reagent, reduces free cytochrome £ with a rate constant dependent on ionic strength, whereas neutral reagents TMPD and DAD were relatively unaffected by ionic strength in their reduction of cytochrome c. The zwitterion cysteine behaved similarly to uncharged reductants DAD and TI~PD in exhibiting only a marginal response to ionic strength. Ascorbate reduces bound cytochrome £ only slowly, but DAD and TMPD reduce bound cytochrome £ rapidly. Reduction of cytochrome £ by DAD and TMPD in the £ - aa3 complex was enhanced lO-fold over DAD reduction of free £ and 4-fold over TMPD reduction of free c. Thus, the importance of ionic strength on the reactivity of cytochrome £ was observed with the general conclusion being that on the cytochrome £ molecule areas for anion (ie. phosphate) binding, ascorbate reduction and complexation to the oxidase overlap. The increased reducibility for bound cytochrome £ by reductants DAD and TMPD supports a suggested conformational change of electrostatically bound c compare.d to free .£. In addition, analysis of electron distribution between cytochromes £ and a in the complex suggest that the midpotential of cytochrome ~ changes with the redox state of the oxidase. Such evidence supports models of the oxidase which suggest interactions within the enzyme (or c - enzyme complex) result in altered midpoint potentials of the redox centers.

One of the most heavily researched proteins in existence, cytochrome c has proved irresistible to chemists and biophysicists for decades. This volume serves as a source book to update the vast body of literature compiled on this protein over the last 40 years. Chapters from an internationally renowned group of experts provide extensive coverage of structural studies, spectroscopic properties, thermodynamic properties, electron transfer kinetics and protein modification. "... a valuable addition to the cytochrome literature; I will certainly get a copy for my group." Dr G. R. Moore, University of East Anglia "For any and all students of the science of cytochrome c, this is an indispensable text." SIM News

Cytochrome c fulfills a central role in biological electron transport. This book draws together information from diverse disciplines in order to provide a common base for further research. The comprehensive treatment of this subject does not neglect to show the diversity of biological respirations and photosyntheses. But it also defines their unifying principles. This overview presents the evolutionary relatedness in bioenergetic systems. Such systems are discussed at the experimental level with emphasis on the interpretation of results and the methodological approaches used. No other text provides a broad survey of this central area of biology. Researchers on cytochrome c are presented with information on the impact and importance of other disciplines on their area of investigation. Advanced students gain a balanced account of biological electron transport and will be encouraged to seek new directions of research.

Cytochromes c are haemoproteins which carry out electron transfer in a wide variety of biological systems, necessitating different kinds of cytochrome c to fulfill different biological roles. The evolutionary relationship between cytochromes c and their host organisms are described, as well as their structural, spectroscopic and redox properties, including both electron-transfer rates and redox potentials. The treatment is aimed at the non-specialist so that both the techniques described and their application to cytochromes c can be understood. All classes of cytochrome c are dealt with to provide a comprehensive account of the field. No other text provides such a broad survey. Similar to the earlier volume "Cytochromes c: Biological Aspects" which deals with the classification, biosynthesis and biological role of cytochromes c, the present book is aimed at research workers and advanced students.