The photoactivated triplet state of metal-free and metal-substituted hemeproteins and model compounds

The photoexcited triplet state of metal-free and closed-shell metal derivatives of horse cytochrome c and horse myoglobin was used to explore chromophore-protein interactions using the technique of electron paramagnetic resonance (EPR). Zero-field splitting (ZFS) parameters, line shape analysis, spin lattice relaxation and Jahn-Teller distortion in the temperature range 3.6-150 K are described. Several conclusions are borne out of this study. (i) For the closed-shell metal derivatives, the ZFS parameters, especially the E value, that assesses in-plane symmetry, has been determined to be sensitive to heme pocket symmetry breaking caused by local electrostatic fields. (ii) These crystal-fields also split the Jahn-Teller susceptible $\rm\sp3E\sb{u}$ triplet state into two vibronic triplet states with an energy splitting that can be correlated to the strength and asymmetry of the internal electric fields within the hydrophobic core of the heme pocket. (iii) The two vibronically split triplet states have been shown to undergo interconversion when $\rm k\sb{B}T$ exceeds the energy splitting of the two vibronic states, in accordance to the theory developed by Hoffman and Ratner (Mol. Phys. 35, 901 (1978)). This dynamic Jahn-Teller Effect manifest itself in the EPR spectrum as a coalescence of the X and Y canonical transitions. The line shape change as a function of temperature can thus be used to probe the extent symmetry breaking of the triplet manifold within a protein environment. (iv) The E values and the order of magnitude energy splittings of the triplet state in the substituted heme proteins are in accord with results that have been obtained from Mossbaurer spectroscopy and from theoretical electric field calculations. (v) For the metal-free derivatives, due to the reduced symmetry of the porphyrin macrocycle, the ZFS parameters are less sensitive to heme pocket interactions. For free-base cytochrome c, the triplet EPR spectrum indicates an axially symmetric environment whereas in protoporphyrin IX substituted myoglobin, a small, but significant E value is obtained indicating greater asymmetry in-plane in the myoglobin heme pocket as compared to cytochrome c. These results are supported by hole burning spectroscopy and by theoretical electric field calculations