Electronic State of the His/Tyr-Ligated Heme of BthA by Mössbauer and DFT Analysis

The BthA protein from the microorganism Burkholderia thailandensis contains two hemes with axial His/OH₂ and His/Tyr coordinations separated by the closest interheme distance of 14 Å. BthA has a similar structure and belongs to the same family of multiheme cytochrome c peroxidases as MauG, which performs long-range oxidation of the partner protein methylamine dehydrogenase. Magnetic Mössbauer spectroscopy of the diferric state of BthA corroborates previous structural work identifying a high-spin (His/OH₂) peroxidatic heme and a low-spin (His/Tyr) electron transfer heme. Unlike MauG, addition of H₂O₂ fully converts the diferric form of BthA to a stable 2e^- oxidized state, allowing a new assessment of this state. The peroxidatic heme is found to be oxidized to a canonical compound II, S = 1 oxoiron(IV) heme. In contrast, the electronic properties of the oxidized His/Tyr heme are puzzling. The isomer shift of the His/Tyr heme (0.17 mm/s) is close to that of the precursor S = 1/2 Fe³⁺ heme (0.21 mm/s) which suggests oxidation of the Tyr. However, the spin-dipolar hyperfine coupling constants are found here to be the same as those for the ferryl peroxidatic heme, indicating that the His/Tyr heme is also a compound II, S = 1 Fe⁴⁺ heme and ruling out oxidation of the Tyr. DFT calculations indicate that the unusually high isomer shift is not attributable to the rare axial His/Tyr heme coordination. The calculations are only compatible with spectroscopy for an unusually long Fe⁴⁺-O_Tyr distance, which is presumably under the influence of the protein environment of the His/Tyr heme moiety in the H₂O₂ oxidized state of the protein. The results offer new insights into how high valence intermediates can be tuned by the protein environment for performing long-range oxidation.