Leghemoglobin: Properties and Reactions

Davies, Michael J.; Mathieu, Christel; Puppo, Alain

doi:10.1016/s0898-8838(08)60154-3

Cited by 17 publications

(14 citation statements)

References 140 publications

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“…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].…”

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confidence: 99%

Investigation of the haem–nicotinate interaction in leghaemoglobin

Patel

Jones

Raven

2000

European Journal of Biochemistry

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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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confidence: 99%

Investigation of the haem–nicotinate interaction in leghaemoglobin

Patel

Jones

Raven

2000

European Journal of Biochemistry

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“…4). This hypothesis is supported by the observation that hydrophobic amino acids located nearby the distal side of the heme pocket partially stabilize ferulic acid in the cyanide-ligated HRP (Veitch, 2004), and that soybean Lba Tyr133, lupin Lb Tyr151 and maize Hbm Tyr151 (an homologous to rice Hb1 Tyr150) might transfer electrons for a pseudoperoxidase activity (Davies et al, 1999;Saenz-Rivera et al, 2004).…”

Section: Comparative Analysis Of the Rice Hb1 And Hrp Heme Pocketmentioning

confidence: 86%

“…Characterizing the peroxidase activity of Hbs is thus of interest because this activity modulates levels of reactive oxygen species (ROS), and thus a variety of cellular processes (Bolwell, 1999;Finkel, 1999;Joo et al, 2001;Rodriguez et al, 2002;Apel and Hirt, 2004;Gapper and Dolan, 2006;Kwak et al, 2006). Evaluation of peroxidase activity from diverse Hbs is well documented (Job et al, 1980;Wan et al, 1998;Davies et al, 1999;Kvist et al, 2007). In plants, peroxidase activities of Arabidopsis thaliana nsHb-1, nsHb-2 and 2/2-like Hbs (AtGLB1, AtGLB2 and AtGLB3, respectively) were reported by Sakamoto et al (2004).…”

Section: Introductionmentioning

confidence: 99%

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

Violante-Mota¹,

Tellechea²,

Morán³

et al. 2010

Phytochemistry

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In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H2O2 was 86-times lower than that of HRP (K(m)=23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor was 2838-times lower than that of HRP (k(cat)/K(m)=15.8 and 44,833 mM(-1) min(-1), respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H2O2 concentration, and that it poorly protects recombinant Escherichia coli from H2O2 stress. These observations indicate that rice Hb1 inefficiently scavenges H2O2 as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.

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“…During pseudoperoxidase reactions of Lbs one electron is transferred from an amino acid sidechain in the Lb heme pocket, generating a cation radical. EPR analysis showed that soybean Lba Y133 and lupin Lb Y138 are candidates for electron transfer [9]. Hbm Y151 is located at a similar distance and orientation from heme-Fe as soybean Lba Y133 and lupin Lb Y138 (Table 1, Fig.…”

Section: Analysis Of Hbm Tertiary Structurementioning

confidence: 96%

Modeling the tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin

Saenz-Rivera

Sarath

Arredondo‐Peter

2004

Plant Physiology and Biochemistry

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Modeling the tertiary structure of a maize (Zea mays ssp. mays) nonsymbiotic hemoglobin" (2004 AbstractThe tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin (Hbm) was modeled using computer tools and the known tertiary structure of rice Hb1 as a template. This method was tested by predicting the tertiary structure of soybean leghemoglobin a (Lba) using rice Hb1 as a template. The tertiary structures of the predicted and native Lba were similar, indicating that our computer methods could reliably predict the tertiary structures of plant Hbs. We next predicted the tertiary structure of Hbm. Hbm appears to have a long pre-helix A and a large CD-loop. The positions of the distal and proximal His are identical in Hbm and rice Hb1, which suggests that heme-Fe is hexacoordinate in Hbm and that the kinetic properties of Hbm and rice Hb1 are expected to be very similar, i.e. that Hbm has a high O 2 -affinity. Thermostability analysis showed that Hbm CD-loop is unstable and may provide mobility to amino acids located at the heme pocket for both ligand binding and stabilization and heme-Fe coordination. Analysis of the C-terminal half of Hbm showed the existence of a pocket-like region (the N/C cavity) where interactions with organic molecules or proteins could be possible. Lys K94 protrudes into the N/C cavity, suggesting that K94 may sense the binding of molecules to the N/C cavity. Thus, it is likely that the instability of the CD-loop and the possibility of binding molecules to the N/C cavity are essential for positioning amino acids in the heme pocket and in regulating Hbm activity and function.

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Leghemoglobin: Properties and Reactions

Cited by 17 publications

References 140 publications

Investigation of the haem–nicotinate interaction in leghaemoglobin

Investigation of the haem–nicotinate interaction in leghaemoglobin

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

Modeling the tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin

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