NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: Relationship to O₂ reactivity

Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O₂ to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires Fe^II for catalysis, but Mn^II can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O₂ and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O₂ through the divalent metal. In O₂ reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient M^III-O₂ ^·- species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the M^III-O₂ ^·- species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O₂ surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O ₂-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of Fe^II and Mn ^II. Owing to the higher potential of Mn, the formation of the NO adduct or the O₂ adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O₂ binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O₂-linked turnover. Accordingly, past studies have shown that intermediate Fe^III species are observed for these mutant enzymes.