Interaction Domain for the Reaction of Cytochrome c with the Radical and the Oxyferryl Heme in Cytochrome c Peroxidase Compound I

Abstract

Site-directed mutants of cytochrome c peroxidase (CcP) were created to modify the interaction domain between CcP and yeast iso-1-cytochrome c (yCC) seen in the crystal structure of the CcP-yCC complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. In the crystalline CcP-yCC complex, two acidic regions of CcP contact lysine residues on yCC. Mutants E32Q, D34N, E35Q, E290N, and E291Q were used to examine the effect of converting individual carboxylate side chains in the acidic regions to amides. The A193F mutant was used to test the effect of introducing a phenyl moiety at the point of closest contact between CcP and yCC in the crystal structure. Stopped-flow experiments carried out in 310 mM ionic strength buffer at pH 7 revealed that yCC initially reduced the indole radical on Trp-191 of the parent CcP compound I with a bimolecular rate constant ka = 2.5 x 10(8) M-1 s-1. A second molecule of yCC subsequently reduced the oxyferryl heme of compound II with a rate constant kb = 5 x 10(7) M-1 s-1. The bimolecular rate constants ka and kb were affected in parallel by each mutation examined. CcP mutants D34N and E290N that are closest to a complementary yCC lysine residue in the crystalline CcP-yCC complex gave the lowest values for ka and kb, which were 25-50% of the values of the CcP parent. Mutants E32Q and E291Q that are removed from the interaction domain gave the same ka and kb values as the CcP parent.(ABSTRACT TRUNCATED AT 250 WORDS)

Constraints on the Radical Cation Center of Cytochrome c Peroxidase for Electron Transfer from Cytochrome c

“…W •+ potentials usually exceed 1 V 45, 81, 90, 91 and most peroxidase Cpd II (Fe(IV)=O) potentials are > 0.9 V 92,93 , yet in WT CcP:Cc, the two-electron couple E o (W •+ Fe(IV)/W o Fe(III)) = ½ [E o (W •+ /W o ) + E o (Fe(IV)/Fe(III))] = 0.740 V. 94, 95 Thus, the protein environment may substantially lower the reduction potentials of the 191 side chain and the porphyrin moiety. 96, 97 Importantly, a lowered potential for W •+ is still consistent with a very small population of the charge-separated intermediate in the WT ZnCcP:Cc system. Provided that the reduction potential of the hopping site remains over ~200 mV higher than that of the donor Cc(II) (E o (Cc) = 290 mV 98, 99 ; i.e ΔG = −200 mV), the standard Marcus equation 42 $(k~k_{eb}(W^{•+})exp[\frac{-{false(\lambda +\mathrm{normal\Delta }Gfalse)}^2}{4\lambda kT}])$ predicts that the back ET rate to a 191 radical will remain ~100× higher than the forward rate of Cc(III) reduction (~ 10 2 s −1 ; Table 2).…”

Role of Configurational Gating in Intracomplex Electron Transfer from Cytochrome c to the Radical Cation in Cytochrome c Peroxidase

“…The effects of these mutations on the interaction with horse and yeast Cc are essentially the same, indicating that both forms of Cc bind to the same site on CcP (30). There is simply a difference in the orientation of hCc and yCc at this binding site as revealed by the Pelletier-Kraut crystal structures (9).…”

Crystal structure of the Leishmania major peroxidase–cytochrome c complex

“…Although this ion pair is right at the interface, it is somewhat surprising that a single mutant has such a large effect because this interaction represents only a small part of the total buried surface area. For example, six different yeast CCP mutants at the interface exhibit between 40-105% of the wild-type control (29) and some of these mutants introduce substantial bulk at the interface.…”