A reaction pathway to compound 0 intermediates in oxy-myoglobin through interactions with hydrogen sulfide and His64

Myoglobin (Mb) binds oxygen with high affinity as a low spin singlet complex and thus functions as an oxygen storage protein. Yet, hybrid Density Functional Theory/Molecular Mechanical (DFT/MM) calculations of oxy-Mb models predict that the O₂ bond is much less resistant to breaking in the presence of hydrogen sulfide (H₂S) compared with water. Specifically, a hydrogen atom from H₂S can be transferred to the distal oxygen atom through homolytic cleavage of the S-H bond to form the intermediate Compound (Cpd) 0 structure and a thiyl radical. In the presence of a neutral His64 (Nε protonation, His64-ε) and H₂S, only a metastable Cpd 0 would be formed as the active site is devoid of any additional proton donor to fully break the O₂ bond. In contrast, the calculations predict that the triplet state is significantly favored over the open shell singlet diradical state throughout the entire reaction coordinate in the presence of H₂S and a positively charged His64. Furthermore, a positively charged His64 can readily donate a proton to Cpd 0 to fully break the O₂ bond resulting in a configuration analogous to reported reaction models of a hemoglobin mutant bound to H₂O₂ with H₂S present. Typically, exotic techniques are required to generate Cpd 0 but under the conditions just described the intermediate is readily detected in UV-Vis spectra at room temperature. The effect is observed as a 2 nm red shift of the Soret band from 414 nm to 416 nm (pH 5.0, His64-εδ) and from 416 nm to 418 nm (pH 6.6, His64-ε).