ELECTRON-PARAMAGNETIC-RESONANCE OF THE EXCITED TRIPLET-STATE OF METAL-FREE AND METAL-SUBSTITUTED CYTOCHROME-C

The photoactivated metastable triplet states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters \D\ for the free-base, Zn and Sn derivatives are 465 x 10(-4), 342 X 10(-4) and 353 x 10(4) cm(-1), respectively, and are temperature invariant over the temperature ranges studied. An \E\ value at 4 K of 73 x 10(-4) cm(-1) was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrin derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the I El value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the \T-2) zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt c are interpreted in terms of vibronic coupling, acid a maximum Jahn-Teller crystal-field splitting of approximately 180 cm(-1) is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The \E\ value is approximately 60 x 10(-4) cm(-1) af 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of greater than or equal to 40 cm(-1) is estimated. In contrast to those for the metal cytochromes, the \E\ value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.