The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the Cbeta of the 2-vinyl group to a methionine residue. To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant). The S160M variant is isolated from Escherichia coli as apo-protein. Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (kcat = 156 +/- 7 s(-1), KM = 102 +/- 15 microM) properties that are analogous to those of rpAPX. The reaction of S160M(R) with H2O2 gives a green protein (S160M(G)). Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160M(G). Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate. The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation. A mechanism that is consistent with the data is presented.

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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...

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