Isolation and preliminary characterization of a soluble nitrate reductase from the sulfate reducing organism Desulfovibrio desulfuricans ATCC 27774

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…With the enzyme in turnover conditions, the resting signal is replaced with a new Mo(V) ion signal (high g nitrate) showing a hyperfine split with a single non-solvent-exchangeable proton (g 1 =1.999, g 2 =1.989, g 3 =1.981, A av =5.1 G), which was suggested to be a catalytic intermediate. This signal is identical to that obtained in D. desulfuricans Nap when reduced samples are reoxidized with nitrate [34]. These preliminary data obtained in Nap from D. desulfuricans and R. sphaeroides suggest that the active sites of the three enzymes are similar.…”

“…EPR and Mo¨ssbauer studies showed the presence of a variable number of Fe/S clusters. All Fdh proteins from SRB exhibit an EPR signal that appears around 70 K and shows no broadening below 40 K, usually named Fe/S I (mean g-values: g 1 =2.048, g 2 =1.947, g 3 =1.890), which is similar to those observed in D. desulfuricans periplasmic nitrate reductase (Nap) [34] and in E. coli Fdh-H [13]. A second broader EPR signal (Fe/S II) appears below 30 K for all the members (mean g-values: g 1 =2.067, g 2 =1.921, g 3 =1.873), and a third broad signal can be discernible in D. alaskensis Fdh below 20 K. D. alaskensis Fdh presents the Mo site magnetically coupled to one of the iron-sulfur clusters, a fact that was never seen before in proteins having a bis-pterin cofactor with a guanine dinucleotide (bis-MGD) coordination.…”

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

“…The cell debris was centrifuged at 16,000g for 30 min. In order to obtain the soluble fraction, the supernatant was ultracentrifuged at 180,000g for 1 h. Purification of Dd NapA up to electrophoretic grade was carried out through a four-step protocol, which has some modifications with respect to the previous ones [15,26]. The soluble extract was loaded into a (diethylamino)ethyl-cellulose column equilibrated with 10 mM Tris-HCl pH 7.6.…”

“…This was concluded from the fact that its maximal expression varies between the species and no transcription promoter or conserved DNA consensus related to the nitrogen metabolism have been found in all the operons that encode them [1]. Therefore, physiological functions such as minimization of the reducing power under certain conditions of growth [7][8][9][10][11][12], denitrification [13][14][15][16], and scavenging nitrate when it is limited in the medium [17] have been proposed for these enzymes. In contrast to most denitrifying bacteria, the sulfate reducer Desulfovibrio desulfuricans ATCC 27774 (Dd) has Nap as the only NR activity.…”

“…In contrast to most denitrifying bacteria, the sulfate reducer Desulfovibrio desulfuricans ATCC 27774 (Dd) has Nap as the only NR activity. This enzyme is expressed when nitrate is used as the final electron acceptor in anaerobic conditions [15], as in the case of Nar from denitrifying organisms. This implies that Nap is used as a respiratory system and, like Nap from Escherichia coli K12 (Ec Nap), nitrate reduction should be coupled to a proton electrochemical gradient generated by using the quinone pool [18,19].…”