Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme

Abstract

The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 μM, V = 9.4 ± 0.5 μM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.

Overexpression, purification, and biochemical and spectroscopic characterization of copper-containing nitrite reductase from Sinorhizobium meliloti 2011. Study of the interaction of the catalytic copper center with nitrite and NO

“…Molecular properties were determined in both cytoplasmic and periplasmic fractions, which were obtained following a procedure reported elsewhere [22]. Protein concentration was determined using the Bradford method with bovine serum albumin as standard [23].…”

The Nitrate Assimilatory Pathway in Sinorhizobium meliloti: Contribution to NO Production

“…Even though narB and nirB are assumed to be part of the nitrate assimilation pathway in S. meliloti no experimental data were available (Luque-Almagro et al, 2011). Ferroni and colleagues measured nitrate reductase total activity in S. meliloti cultures grown under aerobic and microaerobic conditions in the presence of nitrite and/or nitrate (Ferroni et al, 2011). These authors suggested that nitrate reductase activity in aerobic conditions could be associated with assimilatory ammonification, in line with the work of (Sekiguchi and Maruyama, 1988).…”

“…Surprisingly the nitrate assimilation pathway in S. meliloti has received less attention. Eventhough nirBD and narB were suggested to be part of this pathway and biochemical data obtained from crude extracts of S. meliloti indicated the existence of two kinds of nitrate reductase activities (assimilatory and dissimilatory) (Sekiguchi and Maruyama, 1988; Ferroni et al, 2011; Luque-Almagro et al, 2011; Kumar Halder and Chakrabartty, 2015), experimental data were really scarce on the whole. We initiated a study to determine whether NarB and NirB are part of the nitrate assimilatory pathway.…”

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods

“…Kinetic assays using methyl viologen (MV) as electron donor were performed using a discontinuous method adapted from a protocol described previously [42,43]. Solutions Kinetic studies were also carried out by cyclic voltammetry as previously reported but using a 3 mm diameter pyrolytic graphite disk working electrode [21].…”