EFFECTS OF SURFACE AMINO-ACID REPLACEMENTS IN CYTOCHROME-C PEROXIDASE ON INTRACOMPLEX ELECTRON-TRANSFER FROM CYTOCHROME-C

Transient absorption techniques were used to measure the intracomplex electron transfer rates between four recombinant yeast cytochrome c peroxidases and iso-1 cytochrome c (cytc). The binding affinities and catalytic activities with cytc were previously examined [Corin et al. (1991) Biochemistry 30, 11585]. The four include a wild-type peroxidase (ECcP) and three others, each of which has one surface aspartic acid converted to lysine at position 37, 79, or 217. These sites have been suggested to be within or proximal to the recognition site for cytc. These mutants conduct electron transfer with cytc but differ with respect to the ionic strength profiles of their limiting rate constants. At pH and mu = 114 mM, ECcP and D217K show similar limiting rate constants for electron transfer with cytc, k(lim), of ca. 2000 s-1. In the same peroxidase concentration range, the D37K mutant exhibits a k(obs) of ca. 100 s-1. Instability of the compound I form of D79K prevented a complete study of the intracomplex kinetics of this mutant by this technique. At pH 6 and low ionic strength (8 mM), D37K exhibits a dramatic increase in k(obs) to ca. 800 s-1 while the other two recombinants show a marked decrease to values < 150 s-1. D37K displays much lower affinity for cytc than do the other peroxidases at higher ionic strengths [Hake et al. (1992) J. Am. Chem. Soc. 114, 5442], thus preventing adequate complexation necessary for efficient electron transfer. Variations in binding affinity do not explain the more subtle ionic strength kinetic profile observed for D217K. The ''optimal'' ionic strength for this mutant is shifted to higher ionic strength; e.g., at 212 mM D217K exhibits an electron transfer rate constant of 6000 s-1, almost 3-fold that of ECcP under the same conditions. In this case, coupling to the interfacial motions and conformational geometries within the complex must be invoked. Steady-state kinetic measurements conducted with iso-1 cytc yield turnover numbers that are higher than those observed with horse heart cytc but exhibit a similar ordering with D217K > ECcP greater-than-or-equal-to yCcP >> D37K. The maximal turnover obtained from these measurements compare favorably with the limiting rate constants observed from single-turnover kinetics. In general, such experiments help to examine separately the functional roles that specific amino acids play in molecular recognition and electron transfer through the protein matrix.