Narrow Results

In the present work, using the interaction of tetra-(4-sulfophenyl)porphyrin with copper(II) chloride as an example, it has been shown how different amino acid additives (glycine, valine, leucine and tryptophan) catalyze or inhibit the formation of Cu-porphyrin as a function of the chemical environment (borate buffer (pH7.4), DMSO) and the concentration of the additive. It has been demonstrated that the type of amino acid affects the complexation reaction rate. Possible mechanisms of metalloporphyrin formation and the ways of their acceleration are discussed.

As multifunctional catalytic hemoglobins, dehaloperoxidase isoenzymes A and B (DHP A and B) are among the most versatile hemoproteins in terms of activities displayed. The ability of DHP to bind over twenty different substrates in the distal pocket might appear to resemble the promiscuousness of monooxygenase enzymes, yet there are identifiable substrate-specific interactions that can steer the type of oxidation (O-atom vs. electron transfer) that occurs inside the DHP distal pocket. Here, we have investigated the DHP A(V59W) mutant in order to probe the limits of conformational flexibility in the distal pocket as it relates to the genesis of this substrate-dependent activity differentiation. The X-ray crystal structure of the metaquo DHP A(V59W) mutant (PDB 3K3U) and the V59W mutant in complex with fluoride [denoted as DHP A(V59W-F)] (PDB 7MNH) show significant mobility of the tryptophan in the distal pocket, with two parallel conformations having W59-N${}^{𝜀1}$ H-bonded to a heme-bound ligand (H₂O or F${}^{-})$, and another conformation [observed only in DHP A(V59W-F)] that brings W59 sufficiently close to the heme as to preclude axial ligand binding. UV-vis and resonance Raman spectroscopic studies show that DHP A(V59W) is 5-coordinate high spin (5cHS) at pH 5 and 6-coordinate high spin (6cHS) at pH 7, whereas DHP A(V59W-F) is 6cHS from pH 5 to 7. Enzyme assays confirm robust peroxidase activity at pH 5, but complete loss of activity at pH 7. We find no evidence that tryptophan plays a role in the oxidation mechanism ($i.e$. radical formation). Instead, the data reveal a new mechanism of DHP inhibition, namely a shift towards a non-reactive form by OH${}^{-}$ ligation to the heme-Fe that is strongly stabilized (presumably through H-bonding interactions) by the presence of W59 in the distal cavity.