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Flavodoxin from Desulfovibrio vulgaris crystallizes in the oxidized form as well-formed, tetragonal bipyramids, space group P43212, unit-cell parameters, a = b = 51.6 A, c = 139.6 A, 8 molecules per unit cell.The structure has been determined at 2.5-A resolution with phases based on a single isomorphous derivative. The phase ambiguity of a single derivative was resolved by use of anomalous scattering from the single-site Sm+3.The molecule has a five-strand pleated sheet core with two long helices on either side of the sheet. The flavin mononucleotide lies mostly buried on one side of the molecule, but the methyl groups, one edge of the flavin, and part of the ribityl are exposed at the surface.Originally, the name flavodoxin was given by Knight et al.(1) to a small flavoprotein found in extracts of Clostridium pasteurianum grown in iron-deficient medium. The protein was able to replace ferredoxin as an electron carrier (2, 3).After this discovery, other organisms have been shown to contain a similar flavoprotein: Desulfovibrio gigas (4), Peptostreptococcus elsdenii (5), Desulfovibrio vulgaris (6), Clostridium .vIP (7), Rhodospirillum rubrum (8), Chlorella fusca (9), and Escherichia coli (10).Flavodoxins have been defined as low molecular weight proteins with one flavin mononucleotide prosthetic group per molecule and having the ability to function interchangeably with ferredoxin in photosynthetic NADDP+ reduction (11).In Desulfovibrio, flavodoxin can replace ferredoxin in the reduction of sulfite and other sulfur compounds (4, 12, 13), and in the formation of hydrogen from pyruvate via the phosphoroclastic reaction (14).When thiosulfate is reduced by molecular hydrogen in the presence of Desulfovibrio extracts that have been depleted of flavodoxin, ferredoxin, and etochrome cc3, any one of these three electron carriers can couple the reaction (13,15 Sm+3 than for Yb+3, the Sm3+ derivative was used in this work, and to this point only about 50 mg of the protein has been required. Data collectionData were collected on a four-circle, computer-controlled diffractometer with a modified version of the control program of Lenhert and Henry (17). Ni-filtered CuKa radiation was used with a focal spot 0.4 X 10 mm and a take-off angle set at 6°. For both the native crystal and the Sm3+-derivative crystal, data were collected in the same way by a five-step w/20 scan across the top of each reflection. Each step was 0.080 in 20.Backgrounds were not measured for the individual reflections. Instead, the background was determined as a function of 20 by collecting counts at many points between reflections and assuming it to be independent of direction in space. The step scan used to collect data and the method of approximating the background was made necessary by the long c axis.Friedel-related reflections were collected by alternately measuring 32 reflections at +20, followed by 32 reflections at -20. The groups were staggered, however, so that the reflections of each Friedel pair were collected 16 reflections apart.

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Respiratory nitrate reductase purified from the cell membrane of Escherichia coli is composed of three subunits, a, jS, and -y, which are encoded, respectively, by the narG, narH, and narI genes of the narGHJI operon. The product of the narJ gene was deduced previously to be a highly charged, acidic protein which was not found to be associated with any of the purified preparations of the enzyme and which, in studies with putative narJ mutants, did not appear to be absolutely required for formation of the membrane-bound enzyme. To test this latter hypothesis, the narJ gene was disrupted in a plasmid which contained the complete narGHJI operon, and the operon was expressed in a narG::TnlO insertion mutant. The chromosomal copy of the narJ gene of a wild-type strain was also replaced by the disrupted narJ gene. In both cases, when nar operon expression was induced, the a and 0 subunits accumulated in a form which expressed only very low activity with either reduced methyl viologen (MVH) or formate as electron donors, although an a-Is complex separated from the'y subunit is known to catalyze full MVH-linked activity but not the formate-linked activity associated with the membrane-bound complex. The low-activity forms of the a and j8 subunits also accumulated in the absence of the NarJ protein when the 'y subunit (Narl) was provided from a multicopy plasmid, indicating that NarJ is essential for the formation of the active, membrane-bound complex. When both NarJ and NarI were provided from a plasmid in the narJ mutant, fully active, membrane-bound activity was formed. When NarJ only was provided from a plasmid in the narJ mutant, a cytosolic form of the a and , subunits, which expressed significantly increased levels of the MVH-dependent activity, accumulated, and the a subunit appeared to be protected from proteolytic clipping which occurred in the absence of NarJ. We conclude that NarJ is indispensable for the biogenesis of membrane-bound nitrate reductase and is involved either in the maturation of a soluble, active a-" complex or in facilitating the interaction of the complex with the membrane-bound y subunit.

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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 ␣ and ␤ subunits, was purified after release from the membrane fraction by heat treatment, and a detergent-solubilized form, containing ␣, ␤, and ␥ (cytochrome b NR ) subunits, was purified in the presence of Triton X-100. Extraction of soluble ␣␤ 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 ␣␤ dimer. Extraction of the detergent-solubilized ␣␤␥ 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 ␣␤ dimer was estimated to be greater than 6:1. When cytochrome b NR 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 ␣␤ product. These results suggest that menaquinone-9 is specifically bound to the ␣␤ dimer and may be the UV-sensitive component in the pathway of electron transfer catalyzed by nitrate reductase.Nitrate reductase in Escherichia coli is the terminal component of a membrane-bound electron transfer system which catalyzes the reduction of nitrate to nitrite in order to generate energy under anaerobic growth conditions (16). Nitrate reductase, assayed with reduced methyl viologen (MVH) as an artificial electron donor, has been purified by several different procedures, and its chemical and physical properties have been studied in some detail (6,13,23). When purified after solubilization from membranes with detergents, nitrate reductase is composed of three subunits, ␣, ␤, and ␥ (cytochrome b NR ), with approximate molecular masses of 145, 60, and 25 kDa, respectively (5), and contains molybdopterin cofactor (18), several iron-sulfur centers (19), and heme groups (12). An active, soluble form of the enzyme can be released from the membranes in the absence of detergents by heat treatment which, when purified, is composed of only the ␣ and ␤ subunits and contains the molybdopterin cofactor and iron-sulfur centers (19,23,24). Quinols, the presumed physiological electron donors for nitrate reductase, serve as electron donors for nitrate reduction with the detergent-extracted preparation but not with the heat-released enzyme which lacks the cytochrome b NR subunit (28). MVH, which is an effective electron donor for either preparation, apparently donates ...