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

Abstract

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.

Overexpression of phytoglobin in barley alters both compatible and incompatible interactions with the mildew pathogen Blumeria graminis

“…First, the function may be indirect via an effect on the antioxidant system as indicated for both plant and animal systems (Yang et al, 2005;Widmer et al, 2010). Secondly, phytoglobin itself may have peroxidase-like activity, although results on the peroxidase activity of phytoglobin in plants have been inconclusive (Sakamoto et al, 2004;Violante-Mota et al, 2010). However, the authors' unpublished data suggests that peroxidase activity might not be directly attributable to phytoglobin, because peroxidase activity of plant phytoglobin has not been demonstrated in native polyacrylamide gel electrophoresis (data not shown).…”

Plant hemoglobins: a journey from unicellular green algae to vascular plants

“…The rate constants for the reactions of the three Glbs of sugar beet ( Beta vulgaris ) with NO and NO 2 − have been found to be distinctly different, hinting at non‐redundant roles in nitrogen metabolism (Leiva‐Eriksson et al , ). Other reactions of Glbs are the peroxidase activity (Sakamoto et al , ; Violante‐Mota et al , ) and the anaerobic reduction of NO 2 − and NH 2 OH (Sturms et al , ,b). Peroxidase reactions, like reversible ligand binding and the NO‐oxidizing half of the NOD cycle, are a common behavior of Glbs that have been oxidized by peroxides.…”

Synthesis and evaluation of frentizole-based indolyl thiourea analogues as MAO/ABAD inhibitors for Alzheimer’s disease treatment

“…Additionally, an inhibitory effect on HRP was evaluated using modified Ampliflu™ Red, where a direct HRP substrate (H 2 O 2 ) was used instead of in-situ MAO-generated H 2 O 2 . 47,49,59 The assay mixture with a final volume of 200 µL 50 mM potassium phosphate buffer (pH 7.4) in the well contained; Fluorometric determination of the remaining HRP activity was followed by the fluorescence of the resorufin formed (excitation at 535 nm, emission at 595 nm) at 30°C.…”