Direct observation of the ferric-porphyrin cation radical as an intermediate in the phototriggered oxidation of ferric- to ferryl-heme tethered to Ru(bpy)₃ in reconstituted myoglobin

Using semisynthetic myoglobins (Ru(bpy)₃-Mbs) with covalently appended ru(bpy)₃ (bpy = 2,2'-bipyridine), an oxidized Mb is photoproduced through an intramolecular electron abstraction reaction as a key step. UV-vis spectra, electron paramagnetic resonance measurements, and reactivity tests identify the photooxidized Mb as a ferryl species (i.e., Fe⁴⁺-heme). By circular dichroism (CD) spectroscopy, high-performance liquid chromatography (HPLC), and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), it was confirmed that the photooxidation proceeds without damage of the protein structure. Significantly, we report the first direct observation of ferryl-Mb photogeneration via the intermediate porphyrin cation radical. As a consequence of this observation and proposed mechanism, the rate constants for each step can be clearly determined. The photoexcited Ru²⁺(bpy)₃ is oxidatively quenched by [Co(NH₃)₅Cl]²⁺, a sacrificial acceptor, to produce Ru³⁺(bpy)₃, which then proceeds to abstract an electron from the porphyrin ring with a first-order rate constant of 7.1 x 10⁵ s^-1, in the first step. The electron transfer is followed by iron(III) oxidation by the porphyrin radical with concurrent deprotonation (a first-order rate constant of 4.0 x 10⁴ s^-1 at pH 7.5, and 2.0 x 10⁵ s^-1 at pH 9.0) in the second step. Consistent with this mechanism, it is demonstrated that the rate of the fast step of the porphyrin radical generation is independent of pH, whereas the slower step of ferryl-heme formation is dependent on pH. Simulation of the detailed pH dependence of the kinetics clearly shows that the deprotonation-protonation equilibrium of the protein matrix can control the ferryl-heme generation in a heme pocket of Mb.