Investigation of the haem-nicotinate interaction in leghaemoglobin. Role of hydrogen bonding

Abstract

A strategic assessment of the contributions of two active-site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen-bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, K(d) (pH 5.5, μ = 0.10 M, 25.0 ± 0.1 °C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 ± 0.3 μM, 19 ± 1 μM and 11 ± 1μM, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 ± 1 mM, 50 ± 2 mM and 12 ± 1 mM, and for binding of pyridine were 260 ± 50 μM, 4.5 ± 0.5 μM and 66 ± 8 μM, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH-dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pK(a) values 6.9 ± 0.1 and 6.7 ± 0.2, respectively); binding of nicotinate to H61A was independent pH. Reduction potentials for the rLb and rLb-nicotinate derivatives were 29 ± 2 mV (pH 5.40, 25.0 °C, μ =0.10 M) and 65 ± 2 mV (pH 5.42, 25.0 °C, μ=0.10 M), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pK(a) of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.