Control of Formation and Dissociation of the High-Affinity Complex between Cytochrome c and Cytochrome c Peroxidase by Ionic Strength and the Low-Affinity Binding Site

Abstract

A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constants kf and kd of the high-affinity complex between yeast iso-1-cytochrome c (yCc) and cytochrome c peroxidase compound I (CMPI) over a wide range of ionic strength. These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to the cysteine residue in the H39C, C102T variant of yCc, and has the same reactivity with CMPI as native yCc. kd and kf were measured by photoreducing a small concentration of Ru-39-Cc in the presence of the oxidized yCcIII: CMPI complex, which must dissociate before Ru-39-CcII can bind to CMPI and reduce the radical action. The value of kd for the 1:1 high-affinity complex is very small at low ionic strength, < 5 s-1 but is increased significantly by binding yCc to a second low-affinity site. However, the low-affinity yCc binding site is not active in direct electron transfer to either the radical cation or the oxyferryl heme in CMPI, and is too weak to play a role in the kinetics at ionic strengths above 70 mM. The value of kd increases to 4000 s-1 at 150 mM ionic strength, while kf decreases from > 3 x 10(9) M-1 s-1 at low ionic strength to 1.3 x 10(9) M-1 s-1 at 150 mM ionic strength. These studies indicate that the rate-limiting step in enzyme turnover is product dissociation below 150 mM ionic strength and intracomplex electron transfer to the oxyferryl heme at higher ionic strength. The interaction between yCc and CcP is optimized at physiological ionic strength to provide the largest possible complex formation rate constant kf without allowing product dissociation to be rate-limiting. The effects of surface mutations on the kinetics provided evidence that the high-affinity binding site used for the reaction in solution is similar to the one identified in the yCc:CcP crystal structure.

Design of a Ruthenium-Labeled Cytochrome c Derivative to Study Electron Transfer with the Cytochrome bc1 Complex

“…The relative amplitudes of the fast and slow phases are 60 and 40%, respectively, indicating that the 1:1 complex is 40% dissociated, and the equilibrium dissociation constant is K d ) 0.85 µM. The presence of both intracomplex and bimolecular phases at this ionic strength indicates that the bimolecular reaction involves formation of a 1:1 complex followed by intracomplex electron-transfer according to Scheme 2 with k d , k et (43,47). The observed rate constant of the bimolecular phase is given by eq 2:…”

“…where E o is the concentration of cyt bc 1 , and C o is the concentration of Ru z -39-Cc (43,47). From this equation, the formation and dissociation rate constants can be estimated to be k f ) 2.0 × 10 9 M -1 s -1 and k d ) 1.7 × 10 3 s -1 , using the relation K d ) k d /k f .…”

“…These complexes include the R. sphaeroides cyt c 2 :reaction center complex (50), the Cc:cyt c peroxidase complex (35,47,48,51), and the Cc:cyt c oxidase complex (52) in addition to the Cc:cyt bc 1 complex (17). The peripheral electrostatic interactions may guide the docking of Cc to the specific configuration stabilized by the central nonpolar domain that is optimized for rapid electron transfer.…”

Definition of the Interaction Domain for Cytochrome con the Cytochrome bc 1 Complex

“…The presence of both the fast intracomplex phase and the slow bimolecular phase at intermediate ionic strength provides strong evidence that the complete bimolecular reaction involves formation of a 1:1 complex, followed by intracomplex electron transfer according to Scheme 2. The dissociation rate constant k d must be much smaller than k et since if k d were larger than k et , then rapid equilibrium conditions would apply, and separate slow and fast phases would not be observed (31). Assuming that the bimolecular reaction obeys Scheme 2 and k d Ͻ Ͻ k et , the observed rate constant is given by Equation 1 (31),…”

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

“…This observation provides strong evidence that the complete bimolecular reaction between solution Cc and CcO involves formation of a 1:1 complex followed by intracomplex electron transfer according to Scheme 2. The dissociation rate constant k d must be much smaller than k a , since if k d were larger than k a , then rapid equilibrium conditions would apply, and separate slow and fast phases would not be observed (44). Assuming that the bimolecular reaction obeys Scheme 2 and k d Ͻ Ͻ k a , the observed rate constant is given by Equation 2 (44),…”