Abstract Horseradish peroxidase forms spectroscopically distinct, reversible complexes with hydroxamic acids (R─CO─ NHOH), hydrazides (RCO─NHNH2), amides (RCONH2), and α-hydroxyketones (RCO─CH2OH). Binding of these compounds to the enzyme depends on the polar and steric character of R and the hydrogen bonding capacity of ─CO─ X─Y (X─Y = NH─OH, NH─NH2, NH─H, CH2OH). Hydroxamate anions and hydrazide cations do not associate with the enzyme. The dissociation constants (K1) for the enzyme-RCOXY complexes span seven orders of magnitude (K1 ∼ 0.3 to 2 x 10-7 m), the greatest affinity being shown by compounds with a planar, aromatic R group. This is attributed to an interaction of the R moiety at an apoprotein hydrophobic crevice. Spectrophotometric, electron paramagnetic resonance, and magnetic susceptibility measurements indicate that the association of horseradish peroxidase with hydroxamic acids entails a transition from a mixed spin state of the enzyme to a high spin derivative. The spectroscopic characteristics of enzyme-RCOXY complexes are similar, suggesting that X─Y substituents do not interact directly with the metal ion of the prosthetic group but perturb its environment. Analogous conclusions were drawn from (a) the parallelism between RCOXY affinities for manganic and ferric peroxidases which does not pertain to ligands (e.g. F-) substituting in the first coordination sphere of the metal ion and (b) the lack of pronounced spectroscopic changes in RCOXY-ferroperoxidase complexes. The association of peroxidase with hydroxamic acids is competitively inhibited by specific enzyme substrates (hydrogen donors), permitting the evaluation of so far unknown enzyme-(donor) substrate binding parameters (Ks). Such a competitive behavior also implies the proximity of RCOXY to the active site. RCOXY compounds influence heme-linked ionizations and ligand interchange reactions, e.g. they inhibit the formation of alkaline peroxidase and peroxidase-cyanide complex. With cyanide and RCOXY, ferriperoxidase gives tertiary complexes. Their formation occurs sequentially: enzyme (HCN)/⇌ enzyme-cyanide (RCOXY)/⇌ enzyme-cyanide·RCOXY.
Gregory R. Schonbaum (Mon,) studied this question.