From Azotobacter Vinelandii Mutant UW45 (Iron-Molybdenum Cofactor Biosynthss/Nitrogenase/Iron-Sulfur Protein) TIMOTHY D

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6082-6086, August 1989 Biochemistry Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45 (iron-molybdenum cofactor biosynthss/nItrogenase/iron-sulfur protein) TIMOTHY D. PAUSTIAN*t, VINOD K. SHAHtt, AND GARY P. ROBERTS*t§ Departments of *Bacteriology and tBiochemistry and tCenter for the Study of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706 Communicated by Robert H. Burris, May 15, 1989 ABSTRACT The niffl and -E gene products are involved in activity of the newly formed holodinitrogenase is then mea- the synthesis of the iron-molybdenum cofactor of dinitrogen- sured by an acetylene reduction assay in the presence of ase, the enzyme responsible for the reduction of dinitrogen to excess dinitrogenase reductase. ammonia. By using the in vitro iron-molybdenum cofactor The FeMo-co biosynthesis assay provides a system for the biosynthesis assay, we have followed the purification of these detection and isolation of factors and proteins necessary for gene products 450-fold to >95% purity. An overall recovery of FeMo-co synthesis and has been used to identify several 20% was obtained with the purified protein having a specific molecules involved in the process. Homocitrate, the product activity of 6900 units/mg of protein. The protein (hereafter of the NIFV protein, has been found to be essential for the referred to as NIFNE) was found to contain equimolar amounts synthesis of FeMo-co (17). Further work has verified the of the nifN and -E gene products and have a native molecular presence of homocitrate in the finished FeMo-co molecule mass of 200 ± 10 kDa, which indicates an a2.82 structure. (18). The in vitro FeMo-co synthesis assay, along with NIFNE was oxygen labile with a half-life of 1 min in air. A genetic evidence, has also revealed a necessity for dinitro- UV-visible spectrum of the dye-oxidized protein showed an genase reductase during the synthesis of FeMo-co (14, 15, absorption maximum at 425 nm that could be bleached by 18). In this paper we report the utilization ofthis assay for the reduction of NIFNE with sodium dithionite, suggesting the purification of the niJN and -E gene product complex and its presence of an Fe center in NIFNE. characterization. Biological nitrogen fixation is carried out by the bacterial enzyme nitrogenase. Nitrogenase catalyzes the reduction of MATERIALS AND METHODS dinitrogen to ammonia and is composed of two separate Materials. The DEAE-cellulose used was Whatman DE-52 protein complexes, dinitrogenase reductase (also called com- microgranular. The PL-SAX column was obtained from ponent II or iron protein) and dinitrogenase (component I or Polymer Laboratories, Amherst, MA. molybdenum iron protein) (1, 2). Dinitrogenase reductase is Buffer Preparation and Definition. All buffers were titrated a dimer of the nifH gene product with a Mr = 60,000-70,000 to correct pH at room temperature. Anaerobic buffers were (3). It is extremely sensitive to oxygen, contains a 4Fe-4S prepared as described (16). The following abbreviations for center, and is responsible for the reduction of dinitrogenase buffers are used throughout the text. MD buffer is 25 mM (2, 3). Dinitrogenase is an a232 tetramer of the nifK and -D Mops-NaOH (pH 7.4) and 1.7 mM sodium dithionite. MGD gene products and has a Mr of 220,000-240,000 (3). Dinitro- buffer is 25 Mops-NaOH (pH 7.4), 20% glycerol, and 1.7 mM genase is also a metalloenzyme containing Fe, Mo, and S in sodium dithionite. various centers on the protein (4, 5). One ofthese centers, the Bacterial Strains and Growth Conditions. Klebsiella pneu- iron-molybdenum cofactor (FeMo-co), is thought to be the moniae strain UN1217, containing the niflN4536 mutation, an active site for the reduction of nitrogen gas (6-8). The internal deletion of the niJN gene (19), and Azotobacter isolation and characterization of FeMo-co by Shah and Brill vinelandii strain UW45, containing a point mutation in the from purified dinitrogenase showed that the cofactor con- nifB gene (20-22), have been described. Growth and dere- tained Mo, Fe, and S in a ratio of 1:8:6 (4). Subsequent pression ofK. pneumoniae mutants have been described (11, isolation and analysis of FeMo-co by other groups has 23). suggested different stoichiometries of these elements in A. vinelandii cells were grown in modified Burk's minimal FeMo-co, but the consensus remains in the range of 1 medium (24). A. vinelandii strain UW45 was derepressed for Mo:6-8 Fe:8-10 S (3, 4, 8-10). The structure of FeMo-co the nifN and -E gene products, hereafter referred to as remains unknown. NIFNE, as follows. A single colony was inoculated into 100 The products of six nifgenes have been implicated in the ml of Burk's medium plus 28 mM ammonium acetate and synthesis ofthis cofactor in vivo, nifQ, -B, -N, -E, -H, and -V incubated overnight at 30'C. A 2.8-liter flask containing 750 (11-16). Recently, an assay for the in vitro synthesis of ml ofthe same medium was inoculated with 1 ml ofthis starter FeMo-co has been developed (16). This assay involves the culture and incubated in an identical fashion. An 8-liter mixing of extracts of two mutants having lesions in genes carboy containing 6 liters ofthe same medium was inoculated involved in different steps of FeMo-co biosynthesis. Al- with the 750 ml ofculture and incubated with sparging at 30'C though neither mutant is capable of synthesizing FeMo-co overnight. Three hundred liters of Burk's medium was pre- separately, when extracts of appropriate mutants are mixed pared and to this was added 600 ml of filter-sterilized 22% in the presence of an ATP-generating system and molybdate, ammonium acetate to a final concentration of 5.7 mM. The 6 FeMo-co is synthesized. FeMo-co thus synthesized activates liters of overnight UW45 culture was added to bring the FeMo-co-less dinitrogenase present in the extracts. The starting ODwo to 0.050-0.075. The culture was incubated at 30'C and aerated at 140 liters/min with an agitation rate of 150 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: FeMo-co, iron-molybdenum cofactor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 6082 Downloaded by guest on September 24, 2021 Biochemistry: Paustian et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6083 rpm. The presence of ammonium ion was monitored by using linear gradient from 1.0 to 0.0 M (NH4)2SO4 and 0.5-ml Nessler's reagent and after 10-14 hr, ammonium ion was fractions were collected. Pure NIFNE eluted at 0.5 M depleted. The cells were harvested 4 hr after the depletion of (NH4)2S04. ammonia using a Sharples continuous centrifuge. Final OD6w Purified NIFNE was mixed with an equal volume of of the culture was 2.8-3.2. Cell paste was frozen in liquid anaerobic 50%o glycerol in MD buffer to stabilize the activity. nitrogen and stored at -80'C. Typical yields were 950 g ofcell For storage, all fractions were quick frozen in a dry ice/ paste from a 300-liter fermenter. ethanol bath and stored at -20'C. NIFNE stored in this Cell Extract Preparation. K. pneumoniae cell extracts were manner was stable for >3 months. prepared as described except 0.1 M Mops-NaOH (pH 7.4) Oxygen Stability. Twelve hundred fifty units of NIFNE in was substituted for Tris HCI (16). A. vinelandii cell extracts 0.5 ml ofMD buffer was placed into a 9-ml stoppered vial that were prepared by osmotic shock as described (25). had been evacuated and flushed three times with argon and FeMo-co Synthesis Assay. The FeMo-co synthesis assay rinsed with 0.3 ml of MD buffer. The vial was then brought has been described (16, 17). Protein concentrations were to 0.2 atm (1 atm = 101.3 kPa) of oxygen and incubated with determined by the bicinchoninic acid method (26). shaking at 30'C. Samples were taken at 0, 1, 2, 3, 4, 5, and Purification of NIFNE. All steps of the purification were 15 min and were assayed for NIFNE activity. The control carried out under anaerobic conditions at 40C unless other- reaction was carried out in the same manner, without oxygen wise noted. Frozen A. vinelandii UW45 cell paste (240 g) was added. thawed and broken by osmotic shock into 1 liter of MD UV-Visible Scan of N1FNE. Sodium dithionite in the solu- buffer. The lysed cells were centrifuged at 16,000 x g for 50 tion was removed from 4 mg of NIFNE by using a PD10 min at 4TC. The supernatant was applied to a 5 x 25 cm column in an anaerobic glove box. The desalted enzyme (1.3 DEAE-cellulose column that had been reduced with 4 bed ml) was oxidized by mixing with methyl viologen, in its volumes of0.1 M NaCl MD buffer and equilibrated with 1 bed oxidized form, and desalted again with a PD10 column into volume of MGD buffer. After the extract was applied, the 100 mM Mops-NaOH (pH 7.5) to remove the methyl violo- column was washed with 500 ml of MGD buffer followed by gen. The oxidized NIFNE was placed in an anaerobic cuvette 500 ml of 0.11 M NaCl in MGD buffer.

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