Investigation of the haem–nicotinate interaction in leghaemoglobin

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 hydrogenbonding 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, m 0.10 m, 25.0^0.1 8C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4^0.3 mm, 19^1 mm and 11^1 mm, 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 mm, 4.5^0.5 mm and 66^8 mm, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pHdependence 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 of pH. Reduction potentials for the rLb and rLb±nicotinate derivatives were 29^2 mV (pH 5.40, 25.0 8C, m 0.10 m) and 2 65^2 mV (pH 5.42, 25.0 8C, m 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.Keywords: hydrogen bonds; leghaemoglobin; nicotinate; reduction potential.In 1939, Kubo [1] identified a haem-containing pigment, similar to mammalian haemoglobins, in the root nodules of leguminous plants and established that this molecule was capable of reversible oxygenation. The molecule was named leghaemoglobin (Lb) [2] and, since that time, has been the subject of extensive kinetic, spectroscopic and structural investigations [3±6]. The role of Lb is to maintain and regulate the supply of oxygen to respiring Rhizobium bacteria at a level that does not damage the oxygen-sensitive nitrogenase enzyme [3±6]. Isolation of Lb was, in the early days, problematical [7,8], and it was established quite early on that chromatographically pure samples of protein were not similarly homogeneous when examined spectroscopically [7,9]. It was not until 1973 that the nature of the contaminant was found to arise from the interaction of a pyridine 3-carboxylic acid anion (nicotinate, Fig. 1), present under physiological conditions in the root nodules of the plant, with the haem iron [10]. This binding interaction has direct functional consequences, as interaction of a strong ligand with the haem is likely to affect the ability of the molecule to bind dioxygen in an efficient manner [10]. Subsequently, crystallographic (for soybean [11,12] and lupin [13]) and spectroscopic [14] data for the nicotinate derivatives emerged, which confirmed that a deprotonated pyridine nitrogen provides the sixth axial ligand to the haem and which, for soybean Lb, have implicated the participation of the carboxylate group of t...

A simple method for the determination of reduction potentials in heme proteins

“…Similarly, using disodium 2,6-dichlorophenolindophenol ( E m = 217 mV, Table S1), the reduction potential of cytochrome c was determined as E m = 261 mV ± 2 mV (data not shown), which compared well to literature values [34–39]. We find that the methodology is faster than other mediated [23,40–42] methods and more convenient than direct electrochemistry methods [16], because electrons are exchanged in bulk solution instead of via mediated electron transfer between the protein and thin-layer electrode, which means that equilibria are reached more quickly (and avoids very long equilibration times), and the process is not vulnerable to surface contamination. Since the method is based on equilibration, in principle it can be used at any pH or temperature at which the proteins involved are stable and the corresponding equilibration reactions occur at reasonable rates.…”

How is the distal pocket of a heme protein optimized for binding of tryptophan?

“…Ferrous proteins were generated by stoichiometric titration of the ferric enzyme with sodium dithionite. Equilibrium binding constants, K D , were determined in accordance with previously reported procedures .…”

Dinicotinium sulfate

“…The crystal structures of nicotinic acid (Wright & King, 1953;Kutoglu & Scheringer, 1983), dinicotinic acid (Takusagawa et al, 1973), isonicotinic acid (Takusagawa & Shimada, 1976), isonicotinic acid hydrazide (Bhat et al, 1974), nicotinium tetrachlorocuprate(II) (Choi et al, 2002), 2-aminonicotinic acid (Dobson & Gerkin, 1997), 6-aminonicotinic acid hydrochloride (Giantsidis & Turnbull, 2000), nicotinic acid chloride hydrochloride (Nä ttinen & Rissanen, 2003), 3,5-dinitrobenzoic acid nicotinic acid (Zhu & Zheng, 2004), 2-(methylsulfanyl)nicotinic acid (Basavoju et al, 2005), nicotinamide (Wright & King, 1954), 1-methylnicotinamide iodide, chloride and picrate (Freeman & Bugg, 1974), and dinicotinamidium squarate (Bulut et al, 2003) have been reported previously. The crystal structure of nicotinic acid complexed with the protein leghaemoglobin (Ellis et al, 1997) and the haemnicotinate interaction in leghaemoglobin (Patel et al, 2000) were also studied. As part of our investigations of nicotinic acid complexes with inorganic acids, nicotinic acid was treated with sulfuric acid, and the crystal structure of the resulting salt is reported here.…”