On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes

Abstract

We have confirmed the propensity of fragments of cytochrome c to form complexes that reproduce the structure and, in part, the functionality, of the native protein by preparing four novel complexes. We have used trypsin under three different sets of conditions in sequence to prepare a contiguous two-fragment complex (1-55).(56-104). One of the intermediates is a stable overlapping complex (1-65).(56-104). Conditions for limited acid hydrolysis of peptide bonds in cytochrome c have been developed that optimize the yield of fragments (1-50) and (51-104). These two fragments also form a stable association, as do (1-50) and (56-104). These complexes are potentially useful for the semisynthesis of analogues modified in the region of the cleavage sites, which include a number of highly conserved amino acid residues, and are being used for studies of protein folding, interactions with oxidase, cytochrome c immunogenicity and of artificially induced spontaneous resyntheses between complexing fragments. Like other known two-fragment complexes of cytochrome c, they exhibit normal visible spectra, including the presence of the 695 nm band, indicative of a functional haem crevice. Studies of their biological activities and redox potentials lead to a number of conclusions on structure-function relationships in cytochrome c. Most significantly there is a linear relationship between the logarithm of electron-transfer rates from cytochrome c reductase and redox potential in this series of analogues, indicating that such transfer is thermodynamically controlled. This discovery contributes to our understanding of the interaction of cytochrome and reductase. Since the relationship is obeyed by other types of analogues, except for those that involve modification of the active site of cytochrome c, we have a useful diagnostic for those residues that participate directly in electron transfer.

Synergy in Protein Engineering

“…The methionine side chain follows the course of the isoleucine it replaces, but, being unbranched, extends further into the interior of the loop that contains it. In the absence of any other obvious cause, we propose that the small distortion this causes lessens the ability of the loop to shield the bottom of the heme from solvent, and hence lowers redox potential (13,32).…”

“…Mitochondrial Succinate Oxidase Assay-The ability of the mutants to promote electron transfer was assayed using the depleted mitochondria-succinate oxidase assay (13). Rat mitochondria were isolated from hepatocytes by differential centrifugation, and their outer membranes were fractured using osmotic shock, rendering them permeable to proteins.…”

“…While the retained functionality itself implies that such complexes adopt the native fold, in some cases the implication has been verified by x-ray crystallography and spectroscopic techniques (12). Most studies have examined two-fragment noncovalent complexes (13), but examples with three (14) or four fragments (15) are known. One remarkable aspect of these complexes is that the close proximity of the termini at the breakpoints necessitated by the native fold can catalyze the spontaneous reformation of the missing peptide bond(s), in thermodynamically favorable circumstances.…”

Protein Design and the Development of New Therapeutics and Vaccines

“…The chimerae in which both species were vertebrates all exhibited biological activity indistinguishable from that of the parent molecules, providing no support for the "covarion" hypothesis of protein evolution. Study of some vertebrate-yeast chimerae led to the narrowing down of the number of sites likely to be responsible for the functional differences between yeast and vertebrate cytochrome c. Wallace and Proudfoot (1987b) report four novel two-fragment complexes. Two, (1-55)· (56-194) and (1-50)· (51-104), are contiguous, while one, • (56-104), overlaps.…”

Oligomeric oxidation products of the flavonoid quercetin

“…We used K 3 [Fe(CN) 6 ] as an alternate oxidant that is not destroyed in organic media. The redox potential of K 3 [Fe(CN) 6 ] is high and positive, like that of cytochrome c [2,3]. HPLC studies of the reaction products showed that the qualitative compositions of the reaction mixtures obtained using cytochrome c and K 3 [Fe(CN) 6 ] were identical.…”