STUDIES ON STRUCTURE FUNCTION RELATIONSHIPS IN EUCARYOTIC CYTOCHROMES CYTOCHROMES(C)

Methods were developed for the full characterization of cytochrome c derived peptides prior to their use to form modified recombined cytochromes. These peptides included those obtained by limited cyanogen bromide cleavage of native cytochrome c as well as peptides derived from solid phase synthesis.; High performance liquid chromatography (HPLC) on octadecylsilane (ODS) reverse phase columns was shown to be a powerful tool for the characterization of these peptides. Conditions were established for the separation of HP 1-65 (heme peptide containing the first 65 amino acids of cytochrome c) from HP 1-80 and unreacted cytochrome c. Under these same conditions, HP 1-65 with a homoserine lactone at position 65 can be separated from the corresponding homoserine derivative. This lactone is essential to the formation of the covalent bond between residues 65 and 66 in subsequent recombination experiments. HP 1-65 from tuna cytochrome c was shown to be retained more than twice as long as horse cytochrome c HP 1-65 when eluted under identical conditions. This difference can be explained by the lower positive charge of the tuna peptide or independent interaction of the individual amino acids with the column. The three non-heme peptides produced by CNBr cleavage of cytochrome c, NHP 66-104, NHP 81-104, and NHP 66-80, were also easily separated on an ODS column. The extremely high resolution of HPLC was shown with these peptides as NHP 66-104 with a homoserine substituted at position 80 could be separated from the normal Met 80 peptide. NHP 66-80 with the homoserine in the lactone and open forms could also be easily separated. Comparison of the retention of the tuna and horse non-heme peptides showed that the more highly charged tuna peptides eluted first due to decreased hydrophobic interactions with the ODS groups on the packing material.; Unmodified horse, tuna, and bovine cytochromes c were compared with regard to the ionic strength dependence of their redox potentials, ion binding properties, electron transfer kinetics with ascorbate, and biological activity assayed with Keilin-Hartree particles.; The ionic strength dependence of the redox potential of horse cytochrome c showed no evidence of cacodylate binding and gave charges of +6 and +5 on the oxidized and reduced protein, respectively. The tuna and bovine proteins showed deviations from ideal behavior that may have been caused by cacodylate binding so that the charges on the two forms could not be calculated. The ion binding properties of horse cytochrome c were further studied by measuring the effect of Cl('-), H(,2)PO(,4)('-), and Fe(CN)(,6)('4-) on its redox potential. The results showed that all three ions bound to both redox states of this cytochrome.; The rate of reduction of the cytochromes by ascorbate at an ionic strength of 7 mM was measured by stopped-flow techniques and the calculated second order rate constants were: horse--169 M('-1)s('-1), bovine--142 M('-1)s('-1), and tuna--72.8 M('-1)s('-1). The similar rates for the horse and bovine proteins correspond to their nearly identical sequences with the slightly smaller bovine rate being due to the loss of a positive charge at position 60. None of the 18 sequence differences between horse and tuna cytochromes c offer an easy explanation for tuna cytochrome c's significantly lower rate.; The oxidase activities were measured polarographically and all three cytochromes showed two binding sites. The horse and bovine cytochromes showed nearly identical activities while the tuna protein was clearly more active. The increased activity was due to an increased V(,max) as the K(,m)'s for the high affinity site were all approximately 0.038 (mu)M. This indicates that the difference in rate was not due to a difference in binding, but rather to a difference in the actual electron transfer step.