(Oryza Sativa) Recombinant Hemoglobin 1: Implications for in Vivo Function of Hexacoordinate Non-Symbiotic Hemoglobins in Plants
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Phytochemistry 71 (2010) 21–26 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants Fernando Violante-Mota a, Edurne Tellechea b,c, Jose F. Moran c, Gautam Sarath d, Raúl Arredondo-Peter a,* a Laboratorio de Biofísica y Biología Molecular, Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, 62210 Cuernavaca, Morelos, México b FideNa PrincipiaTech Navarra, Edificio CIDEC, C/Tajonar s/n, 31006 Pamplona, Navarra, Spain c Instituto de Agrobiotecnología, Universidad Pública de Navarra-CSIC-Gobierno de Navarra, Campus de Arrosadía, 31006 Pamplona, Navarra, Spain d Grain, Forage and Bioenergy Research Unit, USDA-ARS, East Campus, UNL, Lincoln, NE 68588-0937, USA article info abstract Article history: In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo Received 27 April 2009 as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxi- Received in revised form 7 August 2009 dase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH 6.0 and com- Available online 14 October 2009 pared 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 (Km = 23.3 and 0.27 mM, respectively) and that the catalytic effi- Keywords: ciency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor was 2838-times lower than Rice that of HRP (k /K = 15.8 and 44 833 mMÀ1 minÀ1, respectively). Also, results from this work showed Oryza sativa cat m that rice Hb1 is not chemically modified and binds CO after incubation with high H O concentration, Gramineae 2 2 Function and that it poorly protects recombinant Escherichia coli from H2O2 stress. These observations indicate that Hemoglobin rice Hb1 inefficiently scavenges H2O2 as compared to a typical plant peroxidase, thus indicating that non- Hydrogen peroxide symbiotic Hbs are unlikely to function as peroxidases in planta. Non-symbiotic Ó 2009 Elsevier Ltd. All rights reserved. Peroxidase Plants 1. Introduction has a fraction of hexacoordination which results in moderate O2-affinity (Trevaskis et al., 1997; Smagghe et al., 2009). Non-symbiotic hemoglobins (nsHbs) are O2-binding proteins Transcripts and proteins for nsHb-1 have been detected in nor- that have been identified in primitive and evolved plants, such as mal and stressed plant organs and tissues. For example, nshb-1 bryophytes and angiosperms, respectively. This indicates that genes are expressed in specific tissues from ‘‘normal” plants, such nsHbs are widely distributed in land plants (reviewed by Garro- as the seed aleurone, root meristems and leaf parenchyma (Ross cho-Villegas et al. (2007)). Non-symbiotic Hbs are classified into et al., 2001), and are overexpressed in organs from stressed plants, class 1 and class 2 (nsHb-1 and nsHb-2, respectively). An unique such as those growing under flooding conditions and light and characteristic of the nsHb-1 class is a very high affinity for O2 be- nutrient limitations (Taylor et al., 1994; Lira-Ruan et al., 2001; cause of an extremely low O2-dissociation (koff) rate constant Wang et al., 2003; Ohwaki et al., 2005). These observations suggest (Arredondo-Peter et al., 1997a; Duff et al., 1997; Trevaskis et al., that to nsHb-1 class play specific (non-housekeeping) roles in plant 1997), which results from heme–Fe hexacoordination and ligand cells. However, kinetic properties of nsHb-1 (i.e. high O2-affinity stabilization (Arredondo-Peter et al., 1997a; Hargrove et al., because of a very low koff rate constant) suggest that the nsHb-1 2000; TrentIII et al., 2001). In contrast to nsHb-1, the nsHb-2 class class has functions other than O2-transport (Arredondo-Peter et al., 1998). For example, work using transgenic maize and alfalfa plants overexpressing barley nsHb-1 showed that a probable func- Abbreviations: Hb, hemoglobin; HRP, Horseradish peroxidase; nsHb, non- tion for nsHb-1 was to modulate cellular redox potential and levels symbiotic hemoglobin; PMSF, phenylmethanesulphonyl fluoride; ROS, reactive of NO (Sowa et al., 1998; Dordas et al., 2003, 2004). oxygen species; TMBZ, tetramethylbenzidine. It has also been proposed that the nsHb-1 class could be mul- * Corresponding author. Tel.: +52 777 329 7000x3383; fax: +52 777 329 7040. E-mail address: [email protected] (R. Arredondo-Peter). tifunctional protein and one of the functions could be related to 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.09.016 22 F. Violante-Mota et al. / Phytochemistry 71 (2010) 21–26 pseudoenzymatic activities, such as peroxidase activity (Arredon- 2.2. Analysis of peroxidase activity and kinetic constants of rice Hb1 do-Peter et al., 1998; Saenz-Rivera et al., 2004). Characterizing and HRP the peroxidase activity of Hbs is thus of interest because this activity modulates levels of reactive oxygen species (ROS), and Previously, it was reported that Arabidopsis nsHbs exhibit per- thus a variety of cellular processes (Bolwell, 1999; Finkel, oxidase activity against Amplex Red, DHR123 and guaiacol sub- 1999; Joo et al., 2001; Rodriguez et al., 2002; Apel and Hirt, strates, and that the rate of oxidation for these substrates was 2004; Gapper and Dolan, 2006; Kwak et al., 2006). Evaluation similar (Sakamoto et al., 2004). We evaluated the peroxidase of peroxidase activity from diverse Hbs is well documented activity of recombinant rice Hb1 by using the H2O2/guaiacol as- (Job et al., 1980; Wan et al., 1998; Davies et al., 1999; Kvist say at pH 6.0 and compared it to that from HRP. Initial velocities et al., 2007). In plants, peroxidase activities of Arabidopsis thali- were obtained by measuring the rate of guaiacol oxidation while ana nsHb-1, nsHb-2 and 2/2-like Hbs (AtGLB1, AtGLB2 and varying the H2O2 concentration. Results showed that the initial AtGLB3, respectively) were reported by Sakamoto et al. (2004). velocity for the peroxidase activity of rice Hb1 and HRP obeys These authors showed that AtGLB1, AtGLB2 and AtGLB3 oxidize a Michaelis–Menten equation (Fig. 1A). Steady-state kinetic con- Amplex Red, DHR123 and guaiacol substrates and that rate of stants (Vmax and Km) were obtained after fitting the data to oxidation increased with protein concentration. Also, it was re- Lineweaver–Burk double reciprocals plot (Fig. 1B). Results ported that overexpression of AtGLB1 increased tolerance of showed that the affinity of rice Hb1 for H2O2 was 86-times low- transgenic Arabidopsis to H2O2 stress, which suggested that er than that of HRP (Km = 23.3 and 0.27 mM, respectively), that AtGLB1 plays a role as an antioxidant (Yang et al., 2005). How- rice Hb1 exhibits a very low turnover of H2O2 per unit of time À1 ever, no evidence has been published indicating that peroxida- as compared to HRP (kcat = 368 and 12 105 min , respectively), tive activity from nsHb-1 is of physiological significance. and that the catalytic efficiency of rice Hb1 was 2 838-times À1 À1 Specifically, it is still possible that the effects reported by Yang lower than that of HRP (kcat/Km = 15.8 and 44 833 mM min , et al. (2005) resulted from an indirect activity of AtGLB1 on mit- respectively) (Table 1). These observations suggest that rice igating ROS levels. A direct evaluation of peroxidase activity of Hb1, and probably other nsHb-1, does not function in vivo as a nsHb-1 is thus required, and such kinetic data need to be com- peroxidase. pared to that of a typical plant peroxidase such as horseradish peroxidase (HRP). In this work, we analyzed the peroxidase 2.3. Analysis of effect of H2O2 on structure and ligand-binding activity of rice recombinant Hb1 (a nsHb-1) compared to those properties of rice Hb1 from HRP. Results showed that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor Hemeproteins, including Hbs, react with H2O2 with subsequent is several orders of magnitude lower than that of HRP, suggest- tyrosine–tyrosine cross-linking and oligomerization of the protein. ing that rice Hb1, and most likely other plant nsHb-1, do not For example, the Mycobacterium tuberculosis truncated HbO con- function in vivo as peroxidases. tains six tyrosines which have been postulated to facilitate forma- tion of oligomers after reaction with H2O2 (Ouellet et al., 2007). We examined the effect of incubating rice Hb1 with H2O2 by detecting 2. Results and discussion protein oligomerization and heme modification using SDS–PAGE and visible spectroscopy, respectively (see Section 4.4.). SDS–PAGE The O -binding properties of nsHb-1 suggest that these proteins 2 analysis indicated that no oligomers were formed after incubating do not release O2 after oxygenation because of an extremely low rice Hb1 with 1.2 to 520 mM H2O2 (not shown). Rice Hb1 contains koff rate constant. Thus, it was proposed that the nsHb-1 class only one external tyrosine (Tyr111) potentially available for con- has functions other than O -transport (Arredondo-Peter et al., 2 version to tyrosyl radicals and radical combination. Thus, it is likely 1997a, 1998). For example, it has been proposed that these pro- that rice Hb1 does not form oligomers at high concentrations of teins function in vivo as peroxidases or in some other aspects of H2O2 (i.e. 520 mM H2O2) because of the existence of only one ROS metabolism (Saenz-Rivera et al., 2004; Sakamoto et al., external tyrosine. 2004; Yang et al., 2005).