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Evidence for non-redox quenching of zinc-cytochrome C peroxidase by Fe3+ cytochrome C.
228 Abstracts
CO17 EVIDENCE
FOR NON-REDOX QUENCHING OF ZINC-CYTOCHROME C PEROXIDASE BY FE3+ CYTOCHROME C.
Y.L.
D&Tan,
J.Y.
Nocek,
A.O.
Xeuk,
B.Y.
Hoffman,
Department of Chemistry, Northwestern University, Evanston, Illinois, 60208, U.S.A. A luminescent derivative of cytochrome c peroxidase (CcP) was prepared through reconstitution of apo-CcP with zinc-protoporphyrin IX. [(ZnP)CcP, Fe3+ Cytochrome c] complexes were pre-formed by addition of Fe3+Cytochrome c (Cc) in a 1O:l molar excess over (ZnP)CcP in 5 e phosphate, pH 7.0. Photo-excitation of the ground state (ZnP) was used to produce 3(ZnP), and transient absorption spectroscopy was used to study the subsequent quenching by Fe3+Cc over the temperature range 0-200(C) [ll. Quenching can have several components, among them electron transfer and Fdrster energy transfer 121. We can distinguish between these by making joint measurements of the rate constants for the total quenching (k ) of the photoinitiated '(ZnP) state by Fe3+Cc, and elec1 ron transfer quenching (k,), as inferred from yields of the electron transfer intermediate, [(ZnP)+CcP, Fe2+Ccl. When 3(ZnP) is quenched by Fe3+ Cc (horse), k, and k decrease synchronously with lowered temperature, consistezt with k, = k,. In contrast, when 3(ZnP) is quenched by Fe3+ Cc (iso-11, k, decreases as expected, while k, increases with lowered temperature. This increase in total quenching by the Fe3+Cc (fungal) cytochromes, as electron transfer quenching decreases necessitates the existence of a non-redox mode of quenching, and further indicates k, >> k,. Based on the spectral overlap of the '(ZnP)CcP phosphorescence with the out-of-plane polarized 695 nm band of Fe3+ Cc, we attribute the non-redox quenching seen in yeast-type cytochromes c to Forster energy transfer [3]. The difference in quenching modes between complexes with the horse and yeast-type cytochromes c leads us to infer differences in the porphyrin-porphyrin dihedral angles arising from the relative orientations of the (ZnP)CcP and the cytochromes c. J.M. Nocek, E.D.A. Stemp, M.G. Finnegan, T.I. Koshy, M.K. Johnson, E. Margoliash, A.G.Mauk, M. Smith, and B.M. Hoffman, J. dm. Chem. Sot., 113, 6822-6831 (1991). 2. T. Forster, Discuss. Faraday Sot., 27, 7-17 (1959). 3. J.M. Vanderkooi, P. Glatz, G.V. Woodrow, III, Eur. J. Biochem., 110, 189-196 (1980). 1.
CO17 EVIDENCE
FOR NON-REDOX QUENCHING OF ZINC-CYTOCHROME C PEROXIDASE BY FE3+ CYTOCHROME C.
Y.L.
D&Tan,
J.Y.
Nocek,
A.O.
Xeuk,
B.Y.
Hoffman,
Department of Chemistry, Northwestern University, Evanston, Illinois, 60208, U.S.A. A luminescent derivative of cytochrome c peroxidase (CcP) was prepared through reconstitution of apo-CcP with zinc-protoporphyrin IX. [(ZnP)CcP, Fe3+ Cytochrome c] complexes were pre-formed by addition of Fe3+Cytochrome c (Cc) in a 1O:l molar excess over (ZnP)CcP in 5 e phosphate, pH 7.0. Photo-excitation of the ground state (ZnP) was used to produce 3(ZnP), and transient absorption spectroscopy was used to study the subsequent quenching by Fe3+Cc over the temperature range 0-200(C) [ll. Quenching can have several components, among them electron transfer and Fdrster energy transfer 121. We can distinguish between these by making joint measurements of the rate constants for the total quenching (k ) of the photoinitiated '(ZnP) state by Fe3+Cc, and elec1 ron transfer quenching (k,), as inferred from yields of the electron transfer intermediate, [(ZnP)+CcP, Fe2+Ccl. When 3(ZnP) is quenched by Fe3+ Cc (horse), k, and k decrease synchronously with lowered temperature, consistezt with k, = k,. In contrast, when 3(ZnP) is quenched by Fe3+ Cc (iso-11, k, decreases as expected, while k, increases with lowered temperature. This increase in total quenching by the Fe3+Cc (fungal) cytochromes, as electron transfer quenching decreases necessitates the existence of a non-redox mode of quenching, and further indicates k, >> k,. Based on the spectral overlap of the '(ZnP)CcP phosphorescence with the out-of-plane polarized 695 nm band of Fe3+ Cc, we attribute the non-redox quenching seen in yeast-type cytochromes c to Forster energy transfer [3]. The difference in quenching modes between complexes with the horse and yeast-type cytochromes c leads us to infer differences in the porphyrin-porphyrin dihedral angles arising from the relative orientations of the (ZnP)CcP and the cytochromes c. J.M. Nocek, E.D.A. Stemp, M.G. Finnegan, T.I. Koshy, M.K. Johnson, E. Margoliash, A.G.Mauk, M. Smith, and B.M. Hoffman, J. dm. Chem. Sot., 113, 6822-6831 (1991). 2. T. Forster, Discuss. Faraday Sot., 27, 7-17 (1959). 3. J.M. Vanderkooi, P. Glatz, G.V. Woodrow, III, Eur. J. Biochem., 110, 189-196 (1980). 1.