Isolation and identification of menaquinone-9 from purified nitrate reductase of Escherichia coli.
On the basis of the observation that nitrate reductase from Escherichia coli is sensitive to UV irradiation with an action spectrum indicative of a naphthoquinone (F. Brito and M. Dubourdieu, Biochem. Int. 15:1079-1088, 1987), we extracted and characterized quinone components from two different preparations of purified nitrate reductase. A soluble form of nitrate reductase, composed of alpha and beta subunits, was purified after release from the membrane fraction by heat treatment, and a detergent-solubilized form, containing alpha, beta, and gamma (cytochrome bNR) subunits, was purified in the presence of Triton X-100. Extraction of soluble alpha beta form with chloroform-methanol yielded several UV-absorbing components, which were characterized as menaquinone-9 with an oxidized side chain and further photodestruction products of the menaquinone. The total amount of menaquinone extracted into the organic phase was estimated to be 0.97 mol/mol of alpha beta dimer. Extraction of the detergent-solubilized alpha beta gamma form by a similar procedure yielded two naphthoquinone-like components which were characterized by mass spectrometry as the oxidized forms of menaquinone-9 and demethylmenaquinone-9. In this case, the molar ratio of total naphthoquinone to the alpha beta dimer was estimated to be greater than 6:1. When cytochrome bNR and detergent were eliminated from the detergent-solubilized enzyme by heat treatment and ion-exchange chromatography, only menaquinone-9 could be identified in the organic extract of the active alpha beta product. These results suggest that menaquinone-9 is specifically bound to the alpha beta dimer and may be the UV-sensitive component in the pathway of electron transfer catalyzed by nitrate reductase.
Brito
F
F
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston 77030, USA.
DeMoss
J A
JA
Dubourdieu
M
M
D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology* / Q000528:radiation effects; D006358:Hot Temperature; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D017830:Octoxynol / Q000494:pharmacology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012995:Solubility; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013056:Spectrophotometry, Ultraviolet; D014466:Ultraviolet Rays; D014812:Vitamin K / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D024482:Vitamin K 2 / Q000031:analogs & derivatives*
D024482:Vitamin K 2; D014812:Vitamin K; C073355:menaquinone 9; D017830:Octoxynol; D009565:Nitrate Reductases; D050901:Nitrate Reductase