D. J. D. Nicholas

Bio: D. J. D. Nicholas is an academic researcher from Johns Hopkins University. The author has contributed to research in topics: Nitrate reductase & Molybdenum. The author has an hindex of 8, co-authored 8 publications receiving 885 citations.

[144] Determination of nitrate and nitrite

Abstract: Publisher Summary In phenol-disulfonic acid method for nitrate, the nitration of phenol-2,4-disulfonic acid yields an orange-brown solution. This can be measured on a colorimeter with a blue filter. In brucine method for nitrate, nitration of brucine yields an orange-brown solution which can be determined on a colorimeter with a blue filter. Xylen-l-ol method for nitrate depends on the formation of 5-nitro-2,4-xylen- 1-ol, which is volatile in steam and is distilled into dilute sodium hydroxide with which it forms a red salt. This can be determined colorimetrically with a green filter. Nitrite determination by diazotization and coupling reactions based on the formation of a red AZO compound. This involves, first, the reaction in acid solution of a primary amine such as sulfanilic acid or sulfanilamide with nitrite to form a diazonium salt. The latter is then coupled to an aromatic amine to yield the red AZO dye whose concentration can be determined in a colorimeter.

Diphosphopyridine nucleotide-nitrate reductase from Escherichia coli.

Abstract: Neurospora nitrate reductase which catalyzes the reduction of nitrate to nitrite by reduced triphosphopyridine nucleotide (TPNH) is a metallo-flavoprotein with flavin-adenine dinucleotide (FAD) as the prosthetic group and molybdenum as the metal component (Nason and Evans, 1953; Nicholas and Nason, 1954). A comparable FAD-molybdenum-protein which can utilize reduced diphosphopyridine nucleotide (DPNH) orreducedtriphosphopyridine nucleotide has also been characterized from soybean leaves (Evans and Nason, 1953; Nicholas and Nason, in pre8s). In Escherichia coli, however, the nature of the immediate electron donor as well as other properties of nitrate reductase has been less clear. Yamagata (1938, 1939) found that nitrate reductase in a cell-free preparation of E. coli failed to oxidizeDPNH and was strongly inhibited by cyanide. Subsequently, others, using chemically or enzymatically reduced dyes as the electron source, have reported that the enzyme is a sulfhydryl flavoprotein with an iron component as indicated by light-reversible carbon monoxide inhibition (Egami and Sato, 1947, 1948a, b; Sato and Egami, 1949). Joklik (1950), however, was unable to demonstrate an effect of -SH reagents and carbon monoxide. The earlier statement that cytochrome b is identical with the enzyme has been withdrawn (Sato and Niwa, 1952). It is the purpose of this paper to describe the purification and properties of a DPN-linked nitrate reductase in E. coli and its identification

The Mononuclear Molybdenum Enzymes

Abstract: Molybdenum is the only second-row transition metal required by most living organisms, and is nearly universally distributed in biology. Enzymes containing molybdenum in their active sites have long been recognized,1 and at present over 50 molybdenum-containing enzymes have been purified and biochemically characterized; a great many more gene products have been annotated as putative molybdenum-containing proteins on the basis of genomic and bioinformatic analysis.2 In certain cases, our understanding of the relationship between enzyme structure and function is such that we can speak with confidence as to the detailed nature of the reaction mechanism and, with the availability of high-resolution X-ray crystal structures, the specific means by which transition states are stabilized and reaction rate is accelerated within the friendly confines of the active site. At the same time, our understanding of the biosynthesis of the organic cofactor that accompanies molybdenum (variously called molybdopterin or pyranopterin), the manner in which molybdenum is incorporated into it, and then further modified as necessary prior to insertion into apoprotein has also (in at least some cases) become increasingly well understood. It is now well-established that all molybdenum-containing enzymes other than nitrogenase (in which molybdenum is incorporated into a [MoFe7S9] cluster of the active site) fall into three large and mutually exclusive families, as exemplified by the enzymes xanthine oxidase, sulfite oxidase, and DMSO reductase; these enzymes represent the focus of the present account.3 The structures of the three canonical molybdenum centers in their oxidized Mo(VI) states are shown in Figure 1, along with that for the pyranopterin cofactor. The active sites of members of the xanthine oxidase family have an LMoVIOS-(OH) structure with a square-pyramidal coordination geometry. The apical ligand is a Mo=O ligand, and the equatorial plane has two sulfurs from the enedithiolate side chain of the pyranopterin cofactor, a catalytically labile Mo–OH group, and most frequently a Mo=S. Nonfunctional forms of these enzymes exist in which the equatorial Mo=S is replaced with a second Mo=O; in at least one member of the family the Mo=S is replaced by a Mo=Se, and in others it is replaced by a more complex –S–Cu–S–Cys to give a binuclear center. Members of the sulfite oxidase family have a related LMoVIO2(S–Cys) active site, again square-pyramidal with an apical Mo=O and a bidentate enedithiolate Ligand (L) in the equatorial plane but with a second equatorial Mo=O (rather than Mo–OH) and a cysteine ligand contributed by the protein (rather than a Mo=S) completing the molybdenum coordination sphere. The final family is the most diverse structurally, although all members possess two (rather than just one) equiv of the pyranopterin cofactor and have an L2MoVIY(X) trigonal prismatic coordination geometry. DMSO reductase itself has a catalytically labile Mo=O as Y and a serinate ligand as X completing the metal coordination sphere of oxidized enzyme. Other family members have cysteine (the bacterial Nap periplasmic nitrate reductases), selenocysteine (formate dehydrogenase H), –OH (arsenite oxidase), or aspartate (the NarGHI dissimilatory nitrate reductases) in place of the serine. Some enzymes have S or even Se in place of the Mo=O group. Members of the DMSO reductase family exhibit a general structural homology to members of the aldehyde:ferredoxin oxidoreductase family of tungsten-containing enzymes;4 indeed, the first pyranopterin-containing enzyme to be crystallographically characterized was the tungsten-containing aldehyde:ferredoxin oxidoreductase from Pyrococcus furiosus,5 a fact accounting for why many workers in the field prefer “pyranopterin” (or, perhaps waggishly, “tungstopterin”) to “molybdopterin”. The term pyranopterin will generally be used in the present account. Open in a separate window Figure 1 Active site structures for the three families of mononuclear molybdenum enzymes. The structures shown are, from left to right, for xanthine oxidase, sulfite oxidase, and DMSO reductase. The structure of the pyranopterin cofactor common to all of these enzymes (as well as the tungsten-containing enzymes) is given at the bottom.