A kinetic study of the formation of ordered complexes of ferric cytochrome c fragments.

Abstract

The kinetics of complementation of ferric heme fragments and apofragments of horse heart cytochrome c have been investigated using stopped flow techniques. When changes in fluorescence are monitored, a single phase exhibiting second order kinetics is observed for all cases of productive complexes. The rate constants are independent of the presence of overlapping amino acid sequences in the apofragments but are strongly dependent on the amino acid sequences of the heme fragments. Thus for heme fragment (l-38)H, a rate constant of 2.1 X 10’ M-' s-l is obtained with apofragments (l-104), (23-104), and (56-104). For (l-53)H with (l-104) or (54-104), it is 1.5 X 10’ M-’ s-l. The complementation of (l-25)H with (l-104) or (23-104) results in a rate constant of 3.2 x 10’ M-’ s-l, which is greater by an order of magnitude than those for the other heme fragments. In the presence of 10 mM imidazole, the rate constants dramatically increase to 3.1 x 106, and 2.4 x lo6 M-’ s-l for heme fragments (l-38)H and (l-53)H, respectively, but only by approximately 2-fold for (l25)H. This result is attributed to a change in the axial coordination group of the heme iron atom in fragments (l-38)H and (l-53)H from histidine 33 (or 26) to an imidazole molecule, which in turn results in a major perturbation of the interactions between the heme moiety and its host peptide. When the complementation of the productive complexes is monitored by changes in the Soret absorbance, the data cannot be fit to either first order or second order kinetic equations. However, the results for a nonproductive complex ((l-25)H plus apofragment (39-104)) are identical in both absorbance and fluorescence modes. The data can be fit to an equilibrium process with a rate constant of formation of 1.3 x lo6 M-’ s-l and that of dissociation of 0.95 s-l. Thus, the formation of productive complexes appears to involve a second phase of the reaction which does not occur in the case of the nonproductive complex and which causes no additional fluorescence change but does result in further absorbance changes.