Role of Methionine-230 in Intramolecular Electron-Transfer between the Oxyferryl Heme and Tryptophan-191 in Cytochrome-C Peroxidase Compound-Ii

by Liu, Rui-Qin; Miller, Mark A.; Han, Gye Won; Hahm, Seung; Geren, Lois; Hibdon, Sharon; Kraut, Joseph; Durham, Bill; Millett, Francis

The kinetics of electron transfer from cytochrome c (CC) to yeast cytochrome c peroxidase (CcP) compound I were studied by flash photolysis and stopped-flow spectroscopy. Flash photolysis studies employed horse CC derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis(bipyridine)ruthenium (Ru-CC). Initial electron transfer from Ru-CC reduced the indole radical on Trp-191 of CcP compound I [CMPI(IV,R(.))], producing CMPII(IV,R). This reaction was biphasic for each of several Ru-CC derivatives, with rate constants which varied according to the position of the Ru label. For Ru-27-CC labeled at lysine 27, rate constants of 43 000 and 1600 s(-1) were observed at pH 5.0 in 2 mM acetate. After reduction of the indole radical by Ru-CC, intramolecular electron transfer from Trp-191 to the oxyferryl heme in CMPII(IV,R) was observed, producing CMPII(III,R(.)). The rate constant and extent of this intramolecular electron transfer reaction were independent of both the protein concentration and the Ru-CC derivative employed. The rate constant decreased from 1100 s(-1) at pH 5 to 550 s(-1) at pH 6, while the extent of conversion of CMPII(IV,R) to CMPII(III,R(.)) decreased from 56% at pH 5 to 29% at pH 6. The reaction was not detected at pH 7.0 and above. The pH dependence of the rate and extent of this internal electron transfer reaction paralleled the pH dependence of the rate of bimolecular reduction of CMPII(IV,R) by native horse CC measured by stopped-flow spectroscopy at high ionic strength. The internal electron transfer reaction and the bimolecular reduction of CMPII(IV,R) were also affected in parallel by the substitution of I1e for Met-230. Although the initial reduction of the radical in CMPI(IV,R(.)) by Ru-CC was not affected, the conversion of CMPII(IV,R) to CMPII(III,R(.)) could not be detected in photolysis experiments employing the M230I enzyme. Stopped-flow experiments showed that the rate constants for bimolecular reduction of CMPII(IV,R) to CcP(III,R) by horse CC and yeast iso-1-CC were decreased 12-fold by the M230I mutation, even though this mutation had no effect on the initial bimolecular reduction of the radical in CMPI(IV,R(.)). Crystallographic studies of the M230I mutant ruled out changes in the position of Trp-191 as the basis for this effect, suggesting that the internal electron transfer reaction is influenced by the interaction of Met-230 with the indole radical. The results clearly demonstrate that the internal electron transfer reaction and bimolecular reduction of CMPII(IV,R) by CC are altered in parallel by both pH and the M230I mutation, suggesting that electron transfer from Trp-191 to the oxyferryl iron is an obligatory step in the reduction of CMPII(IV,R). This interpretation predicts that the two intermolecular one-electron transfer reactions that reduce CMPI(IV,R(.)) to CcP(III,R) each proceed via reduction of the Trp-191 radical by CC and is consistent with a mechanism that utilizes a single, unique electron transfer pathway.

Journal
Biochemistry
Volume
33
Issue
29
Year
1994
Start Page
8678-8685
URL
https://dx.doi.org/10.1021/bi00195a008
ISBN/ISSN
1520-4995; 0006-2960
DOI
10.1021/bi00195a008