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

Abstract

Homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of 3,4-dihydroxyphenylacetic acid (HPCA). The enzyme requires FeII for catalysis, but MnII 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(s) 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 MIII-O2•− species not observed during turnover of the wild type FeHPCD. The factors governing formation of the MIII-O2•− species are explored here with EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and 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 O2-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 ability of the enzyme to maintain the ~0.8 V difference in the solution redox potentials of FeII and MnII. Due to the higher potential of Mn, the formation of the NO or O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active site His200. The same non-optimal 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 FeIII species are observed for these mutant enzymes.

Redox Cycling, pH Dependence, and Ligand Effects of Mn(III) in Oxalate Decarboxylase from Bacillus subtilis

“…Turning our attention back to Figure B, we notice a very strong sextet signal in the field range between 600 and 1000 G, centered near 760 G ( g eff ≈ 8.8), for the low-pH oxidized sample. This is the field range where parallel-mode EPR for the non-Kramers high-spin Mn­(III) ion in a reasonably rhombic environment is to be expected. ,− The high-pH sample in Figure B does not show this sextet signal after oxidation but instead exhibits a broad trough in the EPR trace, centered around 700 G ( g eff ≈ 9.5). This signal may perhaps be due to a different ligand environment for Mn­(III) with a distorted parallel-mode spectrum broadening out the distinct 55 Mn hyperfine splitting.…”

Mn(II) Binding and Subsequent Oxidation by the Multicopper Oxidase MnxG Investigated by Electron Paramagnetic Resonance Spectroscopy

“…This signal loss is attributed to either oxidation of the substrate to the 3+ or 4+ oxidation state or coordination of the formerly aqueous Mn­(II) ion in a binding site that induces a significantly larger zero-field splitting (ZFS) interaction, which would broaden the monitored EPR feature possibly beyond detection. ,,, Given these two possibilities, the data (red circles) in Figure were fit using a biphasic kinetic model (red circles) dashed line in Figure . The major component (≈68.7%) had an effective rate constant of k 1obs = 0.205 ± 0.001 s –1 and the minor component (≈31.3%) had k 2obs = 0.019 ± 0.001 s –1 .…”

“…66 This behavior is consistent with an S = 5/2 spin center experiencing a modest positively signed ZFS interaction (D > 1000 MHz) suggesting pseudo-octahedrally coordinated Mn(II) ion. 32 For the signals assigned to the binuclear Mn(II,II) site (blue filled circles, Figure 5B), as the temperature is increased to 40 K, the intensity of the class iii multiplet increases, indicating that the donor spin levels operative in the EPR transition become more populated. 33,41 This behavior is consistent with weak antiferromagnetic coupling between the two ions, i.e., the isotropic-exchange-coupling term J is negative in the Heisenberg−Dirac−van Vleck Hamiltonian (H = −2J S 1 S 2 ).…”

Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site

“…Second-sphere residues are frequently employed to modulate the reactivity and or stability of transient Fe–oxo intermediates generated by non-heme oxidase/oxygenase enzymes. For example, within the active site of 2,3-dioxygenase, a second-sphere interaction with a protonated (positively charged) histidine (H200) has a profound influence on the lifetime of transient intermediates as well as the ultimate product formation (intradiol vs extradiol cleavage). − Thus, it is expected that second-sphere interactions also play a pivotal role in regulating thiol dioxygenase reactivity.…”