[70] Isolation and characterization of various nitrogenases

[70]

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1.5% (w/v), pH 7.2, are harvested at log phase of growth, washed and resuspended in an induction medium containing sodium gluconate, 10 mM; sodium glutamate, 10 mM; KH2PO4, 3 mM; Na-KPO4, 10-50 mM; zwitterionic buffer, 50 mM; MgSO4, 0.4 mM; Fe-citrate, 0.1 mM; and Na2MoO4.2H20, 30/~M, pH 5.5-7.5. The experiments are carried out in 500 to 1000 ml fiat bottom reagent bottles containing 10 to 20 ml of medium and are sealed with rubber sleeve stoppers that are tightly wired shut. By repeated evacuation and filling with argon, the oxygen is removed to a concentration less than 0.005%. The bottles are then filled with a gas mixture of Ar-C2H2-CO2 (89: 10:1) and oxygen injected to the appropriate concentration. A concentration of 0.13% 02 in the gas phase was found to give the best specific rates of C2H~ reduction. The cultures are shaken on rotary shakers (170 rpm) at 27 °. The oxygen concentration should be monitored at approximately 12 hr intervals and if necessary adjusted to the desired concentration. Both oxygen and ethylene are determined by gas chromatography. 11

[70] I s o l a t i o n a n d C h a r a c t e r i z a t i o n Nitrogenases I

of Various

By ROBERT R. EADY Nitrogenase, the enzyme responsible for biological nitrogen fixation, is the subject of intensive international research efforts to establish its structure and mechanism of action. This enzyme, which catalyzes the facile reduction of Nz in a protic environment at ambient temperatures and pressures, is only found in prokaryotes and can be separated into two metalloproteins both of which contain Fe, one of which also contains Mo in addition to Fe. The increasing use of physical techniques, such as electron paramagnetic resonance (EPR), M6ssbauer, and stopped-flow spectroscopy, in these studies has necessitated the development of large-scale purification procedures. The conditions of culture, cell storage and breakage, and extraction procedures can be critical in obtaining active extracts, but a 1 Abbreviations: SDS, sodium dodecyl sulfate; EPR, electron paramagnetic resonance: MES, 2-(N-morpholino)ethanesulfonic acid; DTNB, 5,5-dithiobis(2-nitrobenzoate); Kp, Klebsiella pneumoniae; Av, Azotobacter vinelandii; Ac, Azotobacter chroococcum, Cp, Clostridium pasteurianum; Cv, Chromatium vinosum; Bp, Bacillus polymyxa; Rj, Rhizobium japonicum; Rl, Rhizobium lupini.

METHODS IN ENZYMOLOGY,VOL. 69

Copyright© 1980by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181969-8

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variety of different methods have been successfully developed. Nitrogenase components are usually purified at room temperature and stored frozen in bead form in liquid nitrogen. Procedures for purification of nitrogenase components from Azotobacter vinelandii (Av), Clostridium pasteurianum (Cp), and Rhizobium japonicum bacteroids (Rj) have been described in Vol. XXIV of this series, and more recently the procedures for Bacillus polymyxa (Bp) and Rhodospirillum rubrum (Rr) in Vol. 53. Modifications of the methods described originally have been made which have resulted in increased specific activity, and these will be described here together with procedures for the isolation of nitrogenase components from Klebsiella pneumoniae (Kp), Azotobacter chroococcum (Ac), and the Mo-Fe protein from Chromatium vinosum (Cv). Purification of the unresolved nitrogenase complex of A. vinelandii was described in Vol. XXIV and will not be reiterated. Nomenclature of the Nitrogenase Components The nomenclature used by different workers for the component proteins of nitrogenase is confusing: molybdoferredoxin azofermo, component 1, and Mo-Fe protein have been used for the molbydenum- and iron-containing protein; azoferredoxin azoferm, component 2, and Fe protein used for the Fe-containing protein. Here the general terms MoFe protein and Fe protein will be used, and for the proteins from specific organisms the nomenclature of Eady et al. la will be used. A capital letter is used to denote the genus and a lower case letter the species from which the protein was isolated. The number I refers to the Mo-Fe protein, and the number 2 to the Fe protein. The International Commission on Enzyme Nomenclature has recommended the use of the name nitrogenase as a trivial name for nitric-oxide reductase (EC i.7.99.2). The use of this number for nitrogenase is to be avoided; the assignment is misleading since these enzymes are different proteins, and the class 99.2 is inappropriate for the reaction catalyzed by nitrogenase. General Comments on the Purification and Assay of Nitrogenase A major problem in the purification of nitrogenase components is their extreme sensitivity to oxygen. Short exposure to air results in irreversible inactivation, the half-life of the Fe protein is typically 45 sec, and that of the Mo-Fe protein 10 min even when purified from aerobic organisms. The successful purification of these proteins can only be la R. R. Eady, B. E. Smith, K. A. Cook, and J. R. Postgate, Biochem. J. 128, 655 (1972).

[70]

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achieved if air is rigorously excluded. This is usually done by using buffers sparged with N2 or H2 and which contain approximately I mM NazS204 to scavenge residual 02. Syringe transfer techniques are used to manipulate buffers and nitrogenase components, and column effluents are collected in flasks flushed with an inert gas. The adequacy of the anaerobic technique employed during a purification can be conveniently checked using papers impregnated with a viologen dye. These are prepared by soaking strips of chromatography paper (about I × 15 cm) in 0.25 M Tris-HCl buffer, pH 7.4, containing 1% methyl viologen for 5 min, and then drying. A transient blue color when a few drops of buffer are placed on these papers indicates the presence of residual dithionite. The reduction of the substrate analog C2H2 is a sensitive and convenient assay for nitrogenase during purification, and several procedures for this were described in Vol. XXIV of this series. Since purified nitrogenase components have no known intrinsic enzymic activity, the assay of either the Fe protein or the Mo-Fe protein requires that the complementary protein be available. Maximum activity of the Mo-Fe protein is obtained at a 10- to 20-fold molar excess of the Fe protein. The Fe protein has maximum activity at an optimum ratio of the two components; if this ratio is exceeded, inhibition of activity occurs. To obtain reliable estimates of the activity, a number of assays containing a constant amount of Fe protein are assayed with increasing amounts of Mo-Fe protein. In either type of assay specific activity is calculated on the basis of mg of the protein limiting the assay as nmole of product per minute per milligram of protein. The purification procedures described below result in preparations of nitrogenase components which are homogeneous by the standard analytical techniques of protein chemistry. However, the variation in specific activity and metal content of apparently homogeneous preparations of the Mo-Fe protein isolated from a specific organism indicate contamination by inactive or partially active protein. In all probability, the full potential of catalytic activity of either nitrogenase component from any source has yet to be realised. General Comments on the M e a s u r e m e n t of Protein Concentration Metal and Sulfide Content $2042- interferes with many of the standard analytical methods used in the characterization of Fe-S proteins, and it is not always practicable to remove it by gel filtration. The high absorption coefficient of $2042below 330 nm prevents the use of conventional monitoring equipment to determine the protein profile of eluates from columns used in purification. Provided that diluted samples are aerated on a Vortex mixer for 30 sec

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before analysis, both the Folin and biuret methods are reliable for the estimation nitrogenase proteins. Dry weight measurements have shown that no correction factor is necessary if dried bovine serum albumen is used as a standard, la'2 Fe and Mo estimation by atomic absorption spectroscopy is interfered with by $2042-. In addition, Tris-HC1 and the M o - F e protein interfere with Mo estimation by this m e t h o d ? Colorimetric estimation of Fe with c~,a'-bipyridyl and Mo with toluene-3,4-dithiol on wet-ashed samples of protein is the preferred method of analysis. Sulfide estimation by the methylene blue method is strongly inhibited by $2042- and by unknown products formed by air oxidation. 4 This interference can be overcome by restricting sample volumes to less than 50/xl for proteins containing 1 m M $2042- and by increasing the FeCIz in the assay mixture to 2.3-4.6 /zmole. The use of various volumes of sample to establish the linearity of color development and checking the recovery of sulphide from ferredoxin from C. p a s t e u r i a n u m in the presence of the sample under investigation is recommended. 4 Purification P r o c e d u r e s for N i t r o g e n a s e C o m p o n e n t s from Various Organisms Clostridium p a s t e u r i a n u m

Two methods for the purification of nitrogenase components are in current use, both of which use extracts prepared by autolysis of dried cells. Mortenson described a method 5 using protamine sulfate precipitation and gel chromatography which should be consulted for a description of the manipulative procedures involved. The modified method 6 described below is more reproducible and gives a higher yield of Cpl and Cp2 with increased specific activity. Protamine sulfate is added to the crude extract of 180 gm of dry cells in 50 m M Tris-HC1 buffer, pH 8.5. Cpl and Cp2 are precipitated within the concentration range 1-10% (w/w) of protamine sulfate, related to the initial protein concentration. The precipitated proteins are redissolved by the addition of phosphocellulose (Whatman, P 1 1) at a fivefold excess over protamine sulfate. All subsequent steps use 2 M. G. Yates and K. Planque, Eur. J. Biochem. 60, 467 (1975). 3 H. Dalton, J. A. Morris, M. A. Ward, and L. E. Mortenson, Biochemistry 10, 2066 (1971). 4 J.-S. Chen and L. E. Mortenson, Anal. Biochem. 79, 157 (1977). 5 L. E. Mortenson, Vol. 24, p. 446. 6 W. G. Zumft and L. E. Mortenson, Eur. J. Biochem. 35, 401 (1973).

[70]

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50 mM Tris-HCl buffer, pH 7.5, containing 0.1 M NaCI and 1 mM Na2S204. Cpl and Cp2 are separated by gel filtration in Sephadex G-100. Two 7.5 x 50 cm columns are used at this stage, Cpl is eluted as the first dark brown band, and Cp2 as a second yellowish brown band. Purification of Cpl. Cpl from the Sephadex chromatography step is then concentrated by precipitation with protamine sulfate (0.5-5%, w/w), and when redissolved by phosphocellulose treatment chromatographed on two Sephadex G-200 columns (5 × 50 cm). Although 95% homogeneous at this stage, the specific activity of Cpl is approximately doubled, and a contaminating "'demolybdo" species with an EPR signal at g = 1.94 is removed by DEAE chromatography. One gram amounts of Cpl are applied to a DEAE-cellulose (Whatman, DE 52) column (2.5 × 25 cm) and eluted with a linear NaC1 gradient 0.15-0.3 M in 400 ml of TrisHCI buffer. Cpl is eluted after a yellowish brown band. Purification of Cp2. Cp2 from the first Sephadex G-100 column is concentrated by precipitation with protamine sulfate (1-4%, w/w) and after being redissolved by phosphocellulose treatment rechromatographed on a Sephadex G-100 column (4 × 90 cm). Finally, Cp2 is applied to a DEAE-cellulose column (2.5 x 25 cm) and eluted with a linear gradient of NaCI from 0.2-0.4 M in 500 ml of Tris-HC1 buffer.

C. pasteurianum--Alternative Procedure An alternative procedure 7 utilizes polyethylene glycol to precipitate Cpl and Cp2 from crude extracts and DEAE-cellulose chromatography to separate them. One hundred grams of dried cells are lysed by anaerobic incubation at 30° for 1 hr in 20 mM Tris-HC1 buffer, pH 7.4 (15 ml/gm of dry cells), and the crude extract prepared by centrifuging at 27,000 g for 30 min. The pH is then adjusted to 6 by the addition of 0.1 M MES buffer and solid polyethylene glycol 6000 (Union Carbide Corp.) added to 10% (w/v). The precipitate is removed by centrifuging at 27,000 g for 20 min and discarded. Cpl and Cp2 are precipitated together by the addition of more polyethylene glycol to 30% (w/v) and collected by centrifuging at 27,000 g for 1.5 hr. The precipitate is redissolved in 200 ml of 20 mM Tris-HC1 buffer, pH 7.4, containing 10 mM MgC12, 2 mg of DNase and 4 mg of RNase. Any insoluble material is removed by centrifuging at 15,000 g for 10 min and loading the supernatant onto a DEAE-cellulose (Whatman, DE 52) column (3.5 × 12 cm) equilibrated with 25 mM TrisHCI buffer, pH 7.4, containing 0.15 M NaCl. Hydrogenase is eluted as a brown band by washing the column with 150 ml of this buffer, 0.25 M r M.-Y. W. Tso, T. Ljones, and R. H. Burris,Biochim. Biophys. Acta 267, 600 (1972).

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NITROGEN FIXATION

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NaCI is used to elute Cp I as a dark brown band and 0.4 M NaCI to elute Cp2. One millimolar Na2S204 is included in the buffer used to elute Cp2, which is concentrated approximately threefold using an on-line eluate concentrator (Amincon) and purified immediately as described below. Purification o f Cpl--AIternative Procedure. Cpl from the DEAE-cellulose column is adjusted to pH 6.0 by the addition of 0.1 M MES buffer and solid polyethylene glycol added to 5% (w/v). The precipitate is collected by centrifuging and discarded. Cpl is then precipitated by the addition of more polyethylene glycol to 14% (w/v) and collected by centrifuging at 20000 g for 30 min. The precipitate is redisolved in 20 ml of 50 mM Tris-HCl buffer, pH 8.0, and chromatographed at 4° on a Sephadex G-200 column (5 × 85 cm) equilibrated with the same buffer. Cpl is eluted at 850 ml and concentrated to 10-20 mg protein/ml. Purification of Cp2--Alternative Procedure. Cp2 eluted from the DEAE column is immediately further purified to remove contaminating Cpl by gel filtration on a Sephadex G-100 column (2.5 × 85 cm) equilibrated with 50 mM Tris-HCI buffer, pH 8, containing 1 mM Na2S204. Cpl is eluted in the void volume and Cp2 eluted at 180 ml is concentrated on-line to 10-20 mg protein/ml.

Klebsiella pneumoniae In this procedure la'8 extracts ofK. pneumoniae M5al are prepared by pressure disruption, Kpl and Kp2 are purified by DEAE cellulose chromatography and gel filtration. A modification of the original method TM resulting in higher yields and specific activity is the use of DEAE-cellulose to concentrate Kpl in place of the membrane filtration procedure used earlier. All buffers used in the purification contain 0.1 gm/liter of both dithiothreitol and Na2S204, and, unless stated to the contrary, 25 mM Tris-HCl buffer, pH 7.4, is used. Frozen cells (425 gm) are suspended in 300 ml of buffer and kept on ice. Aliquots (40 ml) are disrupted in a French pressure cell at 15000 psi and a crude extract prepared by centrifuging at 25,000 g for 90 min at 5°. Subsequent steps are carried out at room temperature. An equal volume of a slurry of DEAE-cellulose is added to the crude extract, and after 15 min equilibration the suspension is poured onto a 6.5 × 16 cm column of DEAE-cellulose (Whatman DE 52). After the column has compacted, it is washed with 1 bed volume of buffer. The column is then developed stepwise with 150 ml volumes of 0.15, 0.2, 0.21, 0.22, and 0.25 M NaCI in buffer. Kpl is eluted as a broad 8 B. E. Smith, R. N. F. Thorneley, M. G. Yates, R. R. Eady, and J. R. Postgate, Proc. Int. Syrup. Nitrogen Fixation, 1st, 1974 Vol. 1, p. 150 (1976).

[70]

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dark-brown band after the addition of 0.22 M NaC1. Fractions of 50 ml are collected, and those with a specific activity greater than 500 nmole C2H2 reduced/min/mg of protein are combined for further purification. Kp2 is eluted with 500 ml of 90 mM-MgCI~. Purification of Kpl. Kpl eluted from the DEAE column is diluted twofold with buffer and concentrated by absorption onto a small (4 × 5 cm) column of DEAE-cellulose equilibrated with 25 mM Tris-HCl buffer, pH 8.7, and eluted with 0.3 M NaC1. Kpl is obtained at 20-30 mg protein/ ml and 30 ml aliquots chromatographed on a Sephadex G-200 column (5 × 40 cm) equilibrated with 25 mM Tris-HCl buffer, pH 8.7. Kpl is eluted as a discrete brown band. Several columns are necessary at this stage to treat all the Kpl which is essentially homogenous on disc electrophoresis at this stage. Kpl (i.5 gin) is further purified by chromatography on a DEAE-cellulose column (6.5 × 24 cm) equilibrated with 25 mM Tris-HCI buffer, pH 8.7, and eluted with a linear gradient, i.e., of 30-90 mM MgCI2. Fractions (30 ml) are collected, and although no clear separation of Kpl from other protein is observed, the specific activity is increased considerably. The distribution of fractions of increased activity within the salt gradient is rather variable, and it is sometimes necessary to repeat this step to obtain Kpl of maximum activity. Purification of Kp2. Kp2 from the first DEAE column is diluted fourfold and absorbed onto a small (2.5 × 4 cm) column of DEAEcellulose and eluted with 90 mM MgCI2. Aliquots (30 ml) are chromatographed on Sephadex G-200 equilibrated with buffer containing 50 mM MgCI2. Contaminating Kpl is eluted in the void volume, and Kp2 is eluted as a discrete brownish yellow band. It is necessary to reconcentrate and repeat this step to obtain Kp2 with maximum activity which for unknown reasons is higher than that obtained originally. It has been shown that this last step will remove O2-inactivated Kp2 from the native protein. TM

Azotobacter vinelandii Conditions for growth of A. vinelandii OP (ATCC 13705), the preparation of crude extracts by pressure disruption, and the purification and crystallization of Av I were described a in Volume XXIV. The alternative procedure TM described below allows homogeneous preparations of both Avi and Av2 to be obtained, and Avl to be crystallized reproducibly. Frozen cells are resuspended in 25 mM Tris-HC! buffer, pH 7.4, and 9 R. C. Burns and R. W. F. Hardy, Vol. 24, p. 480. 10V. K. Shah and W. J. Brill,Biochim. Biophys. Acta 305, 455 (1973).

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disrupted by osmotic shock treatment with 3 M glycerol. Crude extract containing 2.5-3 gm of protein is loaded onto a DEAE-cellulose (Whatman DE 52) column (2.5 x 17 cm) equilibrated with 25 mM Tris-HC1 buffer, pH 7.4, containing 0.1 gm/liter of NasSzO4. This buffer is used throughout the purification procedure unless stated to the contrary. The column is successively eluted with 2 bed volumes of buffer, 3 bed volumes of 0.1 M NaC1, 1 bed volume of 0.25 M NaCI to elute Avl, and 1 bed volume of 0.5 M NaCI to elute Av2. Both components are eluted in a volume of approximately 20 ml. Purification ofAvl. Avl eluted from the DEAE column in 0.25 M NaCI is then heat-treated at 52° for 10 min and denatured protein removed by centrifuging at 20,000 g for 10 min. The supernatant, containing Av 1, is concentrated threefold by ultrafiltration using an XM-50 membrane (Amicon), diluted with 14 ml of buffer, and rechromatographed on a DEAE-cellulose column (2.5 × 17 cm) equilibrated with buffer containing 0.15 M NaC1 and 3 gm/liter Na2S204. The column is washed with 2-3 bed volumes of this buffer, and then Avl is eluted with 0.25 M NaCI in a volume of 18.5 ml. Avl is then concentrated to 4 ml by ultrafiltration, diluted sixfold with buffer, and reconcentrated. Needle-shaped crystals of Avl begin to form during the reconcentration. The contents of the ultrafiltration cell are transferred to a serum-capped centrifuge tube and kept at 38 ° for 1 hr before collection of the crystals by centrifuging at 20,000 g for 10 min. The supernatant is then removed and the pellet resuspended in 2-3 volumes of 25 mM Tris-HCl buffer, pH 7.4, containing 42 mM NaCI and centrifuged for 20 min. The pellet of Avl crystals is redissolved in 3 ml of 25 mM Tris-HCl buffer, pH 7.4, containing 0.25 M NaC1. A small amount of insoluble white amorphous material is removed by centrifuging. Additional crystals of Avl can be obtained by reconcentration of the mother liquor and repetition of the process described above. Avl can be obtained crystalline after the heat-treatment step, but lower yields result. The crystallization procedure described here is more reproducible 1°'11 than the original dilution method. 9 Purification of Av2. Av2 eluted from the DEAE column in 0.5 M NaC1 is concentrated to 9 ml by ultrafiltration using a UM20E membrane (Amicon). After a twofold dilution with buffer, it is rechromatographed on a DEAE-cellulose column (2.5 × 18 cm) equilibrated with buffer containing 0.25 M NaCI. The column is washed with 2 bed volumes of this buffer before Av2 is eluted with 0.35 MNaCI. Quantities (10-20 mg) of Av2 are further purified by preparative gel electrophoresis using a Fractophorator (Buchler Instruments, Fort Lee, New Jersey) and a 11 W. A. Bulen, Proc. Int. Symp. Nitrogen Fixation, 1st, 1974 Vol. 1, p. 177 (1976).

[ 70]

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pulsed power supply (Ortec 4100, Ortec Inc., Oak Ridge, Tennessee). A l-cm polyacrylamide stacking gel (6%, w/v) and a 4-cm separating gel (8%, w/v) are used with water cooling. The electrode buffers are 65 mM Tris borate, pH 9, containing 0.3 gm/liter Na2S204, and the eluting buffer used is 65 m M Tris-HCl, pH 7.4, containing 0.1 gm/liter each of Na2S204 and dithiothreitol. The gel is prerun for 4-6 hr at 5 mA (75 V at 100 pulses/sec and a discharge capacitance of 1 ~F). Electrophoresis of Av2 is under these conditions for 10-12 hr when the current is increased to 20 mA (150V at 200 pulses/sec). Av2 is eluted in 1.5-2 ml of buffer.

Azotobacter chroococcum In this procedure 2 extracts of A. chroococcum (NCIB 8003) are prepared by pressure disruption. Acl and Ac2 are separated by DEAE chromatography and purified by gel filtration. Freshly harvested cells (1 kg) are suspended in 500 ml of 75 mM Tris-HC1 buffer, pH 7.8, containing 0.1 gm/liter dithiothreitol and kept on ice. The periodic addition of 40% KOH is necessary to maintain the pH of the suspension which is treated in a French pressure cell at 3000 psi in 40 ml aliquots. After disruption the pH is adjusted to 7.4 and a crude extract prepared by centrifuging at 40,000 g for 30 min under N2 at 5°. Subsequent purification is carried out at room temperature in buffers containing 0.1 g/l of both dithiothreitol and Na2SzO4. Catalytic amounts of RNase and DNase are then added to the crude extract which is incubated under N2 for 30 min before an equal volume of a slurry of DEAE-cellulose in 25 mM Tris-HC1 buffer, pH 7.4, is added. After a further 1 hr incubation the suspension is poured onto a column (10 × 20 cm) of DEAE-cellulose (Whatman DE 52). After the slurry has settled the column is washed with 2 liters of 25 mM Tris HCI buffer, pH 7.4, before Acl and Ac2 are eluted together with 90 mM MgC12, This fraction is then either dialyzed overnight or diluted sixfold with 25 mM Tris-HCl buffer, pH 7.4, before rechromatography on a DEAE-cellulose column (4 × 25 cm). Two columns are used at this stage, and both are eluted with volumes of 100 ml of buffer and then NaC1 solutions from 0.1-0.25 M NaC1 in 0.05 M steps. Acl is eluted in 0.15 M, or more usually 0.2 M NaC1. Ac2 is eluted with 90 mM MgCI2 in a volume of 300 ml. Purification of Acl. Acl eluted from the second DEAE-cellulose column by 0.2 M NaCI (total volume 290 ml) is further purified by gel filtration of 40 ml aliquots on a Sephadex G-200 column (5 x 40 cm) equilibrated with 25 mM Tris-HCl buffer, pH 8, containing 0.1 M MgClz. Ac I eluted from this column is then concentrated on a small (3 × 5 cm) column of DEAE-cellulose and eluted with 90 mM MgC12 in 25 mMTris-

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HCI buffer, pH 7.4. The concentrated material is then chromatographed on a Sephadex G-100 column equilibrated with 25 m M Tris-HC1 buffer, pH 7.4, containing 50 mM MgCI2. To obtain homogeneous preparations of Ac 1, it is usually necessary to repeat the Sephadex G-200 chromatography step. Purification ofAc2. Aliquots (30 ml) of Ac2 eluted in 90 mM MgCIz from the second DEAE column are chromatographed on a Sephadex G100 column (5 × 40 cm) equilibrated with 25 mM Tris-HCl buffer, pH 7.4, containing 50 m M MgCI2. This is repeated up to three times to obtain homogeneous Ac2. A DEAE-cellulose column (2 x 5 cm) equilibrated with 25 m M Tris-HC1 buffer, pH 7.4, is used to concentrate Ac2 (eluted with 90 m M MgCI2) before Sephadex chromatography at each stage.

Chromatium vinosum Of the nitrogenase components of this organism, only Cvl has been purified and partially characterized. TM Chromatium strain D is grown under an atmosphere of 5% CO2 in N2 on a modified Pfennig's medium. Cultures from a 20% inoculum are harvested after approximately 72 hr when levels of acetylene reduction are high. Extracts are prepared by pressure disruption 400 gm of the cell paste are suspended anaerobically in 3 volumes of 20 m M Tris-HCl buffer, pH 8.5, and disrupted at 10,000 psi in a homogenizer. Then 1 M MgCl~ is added to a final concentration of 20 m M based on the volume of buffer added to the cell paste, and the suspension is centrifuged for 30 min at 23,000 g. Additional 1 M MgC12 is then added to give a final concentration of 20% based on the volume of the supernatant. The pH is then adjusted to 7.2 with 1 MTris, and Na2S204 (0.5 mg/ml) is added. Photosynthetic lamellae are then precipitated by the addition of 50% (w/v) polyethylene glycol 6000 to a final concentration of 8% (v/v) and removed by centrifuging at 23,000 g for 30 min. Protamine sulfate (50 mg/ml) is added to the supernatant to precipitate nitrogenase. During this process the nitrogenase activity of the supernatant is monitored to establish the concentration range over which precipitation occurs--usually 5-15%. Precipitated material is collected by centrifuging at 23,000 g for l0 min and resolubilized by stirring for 30 min with a sevenfold excess of cellulose phosphate in 20 mM Tris-HCl buffer, pH 8. The supernatant is then diluted to 500 ml with 20 mM Tris HCI buffer, pH 7.5, containing 0.1 M MgCl~. Cvl and Cv2 are then separated by chromatography on a DEAE-cellulose column (4.5 × 25 cm) equilibrated with the same buffer. Cv 1 is eluted with 200 ml of buffer 12 M. W. C. Evans, A. Te|fer, and R. V. Smith, Biochim. Biophys. Acta 310, 344 (1973).

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containing 0.25 M NaCI and Cv2 with 0.6 M NaCl. Cvl is well resolved at this stage but Cv2 contains residual Cvl and ferredoxin. Purification o f Cvl. Cvl from the DEAE-cellulose column is concentrated by ultrafiltration using an XM-100 membrane (Amicon) and chromatographed on a Sephadex G-200 column (4.5 x 40 cm) equilibrated with 40 mM Tris-HCl, buffer, pH 8, containing 0.1 M NaCl. Complete purification requires that this step is repeated.

Criteria of Purity The most widely used test of the purity of nitrogenase components are disc electrophoresis of the native proteins under anaerobic conditions and of the denatured proteins in the presence of SDS. The Mo-Fe proteins when purified by the methods described above are often homogeneous by these criteria before the maximum specific activity has been obtained, suggesting a considerable degree of contamination with inactive protein. The successful removal of a "demolybdo" species of the Mo-Fe proteins is indicated6 by the absence of an EPR signal of the g = 1.94 type in the Na2S204-reduced protein at temperatures below 30° K. Although the correlation between metal content and specific activity is not direct, la in general the Mo and Fe content increase with specific activity to 2 Mo atoms and 24-32 Fe atoms/mole in those of the highest activity. The yields and specific activities of nitrogenase components purified by the procedures described above are shown in Table I.

Properties of the M o - F e Proteins The tendency of these proteins to aggregate (with no loss in activity) on storage in liquid nitrogen, and to dissociate at high dilutions undoubtedly account for the range of values that appeared for the molecular weight in the earlier literature. Within the last five years a number of different techniques have been used to determine molecular weights which now, in most instances, fall in the range of 200,000-235,000 (see Table II). Because of the comparative ease of maintaining anaerobic conditions, gel filtration has been the method most widely used. The subunit structure of purified Mo-Fe proteins determined by electrophoresis in polyacrylamide gels containing SDS 14 is consistent with is W. H. Orme-Johnsonand L. C. Davis, in "'Iron SulfurProteins" (W. Lovenberg,ed.), Vol. 3, p. 15, AcademicPress, New York, 1977. 14K. Weberand M. Osborn, J. Biol. Chem. 244, 4406 (1969).

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TABLE I YIELD AND SPECIFIC ACTIVITIES OF Mo-FE PROTEIN AND FE PROTEIN PURIFIED FROM DIFFERENT ORGANISMS

Mo-Fe protein

Organism C. pasteurianum K. pneumoniae A. vinelandii A. chroococcum C. vinosurn

Fe protein

Specific activity Yield (nmole product/min/mg) ( g m ) >2000 2500 2250 1638 1444 2072 1600

1-2 0.24 0.8 0.048 0.378 1 0.5

Specific activity (nmole product/min/mg)

Yield (gm)

>2200 3100 1200 1815 -2100 __

0.5a 0.18~ 0.128c'a 0.031e --~ 0.73g h

a W. G. Zumfl and L. E. Mortenson, Cur. J. Biochem. 35, 401 (1973). b M.-Y. W. Tso, T. Ljones, and R. H. Burris, Biochim. Biophys. Acta 267,600 (1972). c R. R. Eady, B. E. Smith, K. A. Cook, and J. R. Postgate, Biochem. J. 128, 655 (1972). a B. E. Smith, R. N. F. Thorneley, M. G. Yates, R. R. Eady, and J. R. Postgate, Proc. Int. Syrup. Nitrogen Fixation, 1974 1st Vol. 1, p. 150 (1976). e V. K. Shah and W. J. Brill, Biochim. Biophys. Acta 305, 445 (1973). R. C. Burns and R. W. F. Hardy, Vol. 24, p. 480. M. G. Yates and K. Planque, Cur. J. Biochem. 60, 467 (1975). h M. C. W. Evans, A. Telfer, and R. V. Smith, Biochirn. Biophys. Acta 310, 344 (1973).

the native proteins being tetrameric with a molecular weight near 220,000, but are often contradictory as to the number of types of subunit. 15 Both one or two types of subunit present in equal numbers were reported for various Mo-Fe proteins, and for Avl, conflicting data appeared from different laboratories. It has recently been shown TM that the number of s u b u n i t b a n d s o b t a i n e d with K p l , A c l , A v l , a n d R j l u s i n g this m e t h o d d e p e n d s o n the c o m m e r c i a l s o u r c e o f the SDS u s e d in the e l e c t r o d e buffers. O n e or two d i s c r e t e w e l l - r e s o l v e d b a n d s are o b t a i n e d with diff e r e n t S D S b r a n d s (Fig. l), e m p h a s i z i n g the u n r e l i a b i l i t y of this m e t h o d for d e t e r m i n i n g the s u b u n i t s t r u c t u r e of t h e s e p r o t e i n s . T h e m o l e c u l a r weights n e a r 50,000 a n d 60,000 o b t a i n e d for the s u b u n i t s of C p l agree well with the v a l u e s of 52,500 a n d 62,000 f r o m gel filtration in S e p h a r o s e 6B e q u i l i b r a t e d with 6 M g u a n i d i n e HCI. T h e s u b u n i t s of C p l , K p l , a n d is R. R. Eady and B. E. Smith, in "Dinitrogen Fixation" (R. W. F. Hardy, F. Bottomley, and R. C. Burns, ed.), Vol. 2, p. 399 (Wiley (Interscience), New York, 1979. 18C. Kennedy, R. R. Eady, E. Kondorosi, and D. Klavans Rekosh, Biochem. J. 155, 383 (1976).

[70]

NITROGENASE

765

+. £,1

Ti~ ~ ++

+. t",t

m O r~

[~

o

(-q (-q (,~

,4

e~ ul J

m~

i

=

+

%

! ' -

o

°~C

II

e~ m

O

r~

G

,< ,.4 m O

.-

c~ ' ~

0

-~'~ o&'=

~.~ ~

N

>,.-

o ~

,.

~:

,-,

• ~.=

"~"~-

~-..4

:_o~

.

766

NITROGENFIXATION

a

[70]

b

!

b

Kpl Acl

Avl Rjl

Kpl Acl ~Avl Rjl

Kpl Acl Avl Rjl

FIG. 1. Banding pattern of various Mo-Fe proteins on 10% acrylamide gels using three different brands of SDS. The gels and sample buffer contained Serva brand SDS, the electrode buffer contained SDS from (a) Schwartz, (b) Serva, or (c) Koch-Light. [After C. Kennedy, R. R. Eady, E. Kondorosi, and D. Klavans Rekosh, Biochem. J. 155, 383 ( 1976).]

AvI have been isolated. Those of Cpl by repetitive gel filtration in Sephadex G-100 equilibrated with 20 mM HCI and 0.1%/3-mercaptoethanol, 17 Kpl by preparative SDS electrophoresis, TM and Avl by chromatography on carboxymethyl cellulose equilibrated with 7 M urea at pH 4.6. TM Unfortunately these conditions all result in the loss of Mo and Fe from the subunits, lr The isolation and properties of a low molecular weight O2-sensitive Mo-containing cofactor from Mo-Fe proteins is described by Shah (this volume, Article [72]). The nonidentity of the subunits of Kpl has been established by comparison of peptide maps of tryptic digests of the separated subunits. The amino acid compositions of subunits of a given Mo-Fe protein are very similar, and it is difficult to distinguish between them on this basis.16 The biochemical data are consistent with the M o - F e proteins being tetramers of the o~2~2type. Support for this formulation is provided by a genetical analysis of the nif genes of K. pneumoniae where two separate genes determine the synthesis of Kpl subunit polypeptides. 19 The Mo, Fe, and acid-labile sulfide content of several Mo-Fe proteins are shown in Table III. The correlation between metal content and activity is not directX3; presumably loss of activity can occur without loss of metal. Preparations of M o - F e proteins with the highest specific activity

17 T. C. Huang, W. G. Zumft, and L. E. Mortenson, J. Bacteriol. 113, 884 (1973). is R. H. Swisher, M. L. Landt, and R. J. Reithel, Biochem. J. 163, 427 (1977). 19 R. A. Dixon, C. Kennedy, A. Kondorosi, V. Krishnapillai, and M. J. Merrick, Molec. Gen. Genet. 157, 189 (1977).

[70]

NITROGENASE

767

T A B L E III MOLYBENUM, IRON AND ACID-LABILE SULFIDE CONTENT OF HIGHLY PURIFIED M o - F E PROTEINSa

Protein Cpl

Kpl Avl

Acl Rj I Cvl

Specific activity (nmole product/min/mg)

Mo (gm atom/mole)

1100 1200 2250 2500 1200 2150 1100 1400 1700 2000 1000 1600

1.38 1.3 2 1-1.5 1 2.01 +-- 0.3 1.54 1.6 1.88 1.9 --- 0.3 1.3 1.4

Fe S 2(gm atom/mole) (gm atom/mole) 19.3 20.5 24 12-18 17.5 --_ 0.7 32.5 ± 3 24 28-31 24 22 ± 2 28.8 19

22 b c 24 a 8-15 e 16.7 _ Is __._u 20~ 21-335 2T 22 ± 2k 26.2 h 15m

a Data from refs b, h, and 1 have recalculated on the basis of a M W of 220,000. H. Dalton, J. A. Morris, M. A. Ward, and L. E. Mortenson, Biochemistry 10, 2066 (1971). c j. p. V a n d e c a s t e e l e and R. H. Burris, J. Bacteriol. 101, 794 (1970). d T. C. H u a n g , W. G. Zumft, and L. E. M o r t e n s o n , J. Bacteriol. 113, 884 (1973). e M.-Y. W. Tso, Arch. Microbiol. 99, 71 (1974). s R. R. Eady, B. E. Smith, K. A. Cook, and J. R. Postgate, Biochem. J. 128, 655 (1972). g B. E. Smith, R. N. F. Thorneley, M. G. Yates, R. R. Eady, and J. R. Postgate, Proc. Int. Syrup. Nitrogen Fixation, 1st, 1974 Vol. I, p. 150 (1976). h D. Kleiner and C. H. Chen, Arch. Microbiol. 98, 100 (1974). R. C. Burns and R. W. F. Hardy, Vol. 24, p. 480. J W. A. Bulen, Proc. Int. Symp. Nitrogen Fixation, 1st, 1974 Vol. 1, p. 177 (1976). M. G. Yates and K. Planque, Eur. J. Biochem. 60, 467 (1975). t D. W. Israel, R. L. H o w a r d , H. J. Evans, and S. A. Russell, J. Biol. Chem. 249, 500 (1974). ,n M. C. W. E v a n s , A. Telfer, and R. V. Smith, Biochim. Biophys. Acta 310, 344 (1973).

contain 2 Mo and 22-32 Fe atoms and an approximately equivalent number of acid-labile sulfide atoms per molecule. M6ssbauer and EPR studies often require the use of protein enriched with 5rFe or aSMo. The successful exchange of these metals from Mo-Fe protein has not been reported, and isotopic substitution is achieved by growing the organism on purified medium enriched in the relevant metal isotope. The amino acid composition of purified Mo-Fe proteins are very similar. 15 All the common amino acids are present and acidic residues at 19.4-21.6% are approximately twice as abundant as basic residues. C-

768

NITROGEN FIXATION

[70]

terminal amino acids are alanine and leucine in Cpl z° and serine in Avi, Is and the N-terminal residues in RII are serine, zl Concentrated solutions of Mo-Fe protein are dark brown but the visible absorption spectra are very broad. Features at 525 and 557 nm in some crystalline preparations of Av i have been attributed to cytochrome contamination. 1° Avl is the only Mo-Fe protein which has been crystallized. The procedure is based on the insolubility of this protein at low salt concentrations at neutral pHZ2---a property which is also used in its purification. 9"1° Two types of crystals have been obtained, neither of which is a suitable form for X-ray structure analysis. When the ionic strength is lowered by the rapid dilution of concentrated protein solutions with buffer, small crystals (1-4 × 30-60/zm) are formed, 9"1°the optimum concentration of NaC1 for crystallization is 40-80 mM. Membrane concentration of solutions to saturation results in the formation of aggregated forms ~a (1-3 mm × 40 /xm). Crystallization is not a good criterion of purity TM and Kp i, Cp 1, and Ac I do not crystallize under these conditions. The only spectroscopic technique successfully used to probe the environment of Mo in the Mo-Fe protein is the recently developed technique of X-ray absorption edge spectroscopy. Comparison of the K Xray absorption edge of Cpl with a range of Mo-containing model compounds enable a coordination charge of 2.3 _ .03 to be calculated?a The assignment of Mo in Cp I to a pair of antiferromagnetically coupled Mo(V) atoms has been criticized, z4 and more recent experiments with $2042-reduced Cpl are consistent with the ligand environments of the Mo involving sulfur atoms. 25 At temperatures below 30°K the S2042--reduced proteins exhibit a characteristic EPR spectrum with g values near 4.3, 3.7, and 2.01 (see Table IV and Fig. 2). This type of signal is not shown by any other class of FeS protein and has been assigned to transitions in the S = ½ ground state of spin S = ~ system, z6-2s Kpl and Avl substituted with 57Fe show 2o J.-S. Chen, J. S. Multani, and L. E. Mortenson, Biochim. Biophys. Acta 310, 51 (1973). 21 M. J. Whiting and M. J. Dilworth, Biochim. Biophys. Acta 371,337 (1974). 22 R. C. Burns, R. D. Holsten, and R. W. F. Hardy, Biochem. Biophys. Res. Commun. 39, 90 (1970). 2s S. P. Cramer, R. K. Eccles, F. W. Kutzler, K. O. Hodgson, and L. E. Mortenson, J. Am. Chem. Soc. 98, 1287 (1976). 24 B. E. Smith, J. Less-Common Met. 54, 465 (1977). 25 S. P. Cramer, K. O. Hodgson, W. O. Gillum, and L. E. Mortenson, J. Amer. Chem. Soc. 100, 4630 (1978). 26 B. E. Smith, D. J. Lowe, and R. C. Bray, Biochem. J. 130, 641 (1972). z7 G. Palmer, J.-S. Multani, W. G. Zumft, and L. E. Mortenson, Arch. Biochem. Biophys. 153, 325 (1972). ~8 E. Munck, H. Rhodes, W. H. Orme-Johnson, L. C. Davis, W. J. Brill, and V. K. Shah, Biochim. Biophys. Acta 400, 32 (1975).

[ 70]

NITROGENASE

769

T A B L E IV EPR SIGNALS ASSOCIATED WITH 82042-REDUCED MO-FE PROTEIN AND SIGNALS OBSERVED UNDER DIFFERENT CONDITIONS IN THE STEADY STATE Turnover under

S2OaZ--reduced Kpl

Acl Avl Cpl Bpl Rrl

Rjl Cvl

4.27 3.73 2.018 high pH a 4.32 3.63 2.009 low pH a

4.29 3.65 2.013 b 4.30 3.67 2.01 c 4.27 3.78 2.01 a 4,37 3.53 2.01 ~ 4.34 3.65 2.01 e 4.17 3.73 2.03 f 4.3 3.68 2.01 g

Ar or N2 2.139 2.001 1.977 h 2.092 1.974 1.933 h 2.125 2.000 2.00 n'i 5.7 5.4 h 2.14 2.001 1.976 J 2.09 2.01 1.98 e 2.13 1.99 1.98 e --2.14 2.01 1.98 e --

low CO (0.001 atm)

high CO (0.1 atm)

2.073 1.969 1.927 n 2.17 2.06 2.06 h

2.08 1.97 1.92j 2.01 1.98 1.93 e 2.07 1.975 1.92 e -2.08 1.975 1.93 e ---

2.16 2.07 2.04 J 2.17 2.08 2.05 e 2.15 2.07 2.04 e -2.17 2.09 2.05 e 2.18 2.11 2.06 e --

a B. E. Smith, D. J. Lowe, and R. C. Bray, Biochem. J. 135, 331 (1973). b M. G. Yates and K. Planque, Eur. J. Biochem. 60, 467 (1975). R. C. Burns, R. D. Holsten, and R. W. F. Hardy, Biochem. Biophys. Res. Commun. 39, 90 (1970). d G. Palmer, J. S. Multani, W. C. Cretney, W. G. Zumft, and L. E. Mortenson, Arch. Biochem. Biophys. 153, 325 (1972). e L. C. Davis, M. T. Henzl, R. H. Burris, and W. H. Orme-Johnson, Biochemistry (in press). f H. J. Evans, P. E. Eishop, and D. Israel, Proc. Int. Syrup. Nitrogen Fixation, 1st 1974 Vol. 1, p. 234 (1976). o M. C. W. Evans, A. Telfer, and R. V. Smith, Biochim. Biophys. Acta 310, 344 (1973). h D. J. Lowe, R, R. Eady, and R. N. F. Thorneley, Biochem. J. 173, 277, (1978). l Only observed in the presence of added ethylene. J M. G. Yates and D. J. Lowe, FEBS Lett. 72, 121 (1976). W. H. Orme-Johnson and L. C. Davis, in " I r o n - S u l p h u r Proteins" (W. Lovenberg, ed.), Vol. 3, p. 15. Academic Press, New York, 1977.

about a 5-G linewidth broadening which is not observed when the proteins are substituted with asMo.2e'2r Correlation with M6ssbauer data for these proteins (see below) indicate that this signal is associated with Fe atoms, probably in two F e 4 S 4 clusters. The g values and linewidth of the EPR spectrum of Kpl are pH dependent 2a and the interconversion of the high and low pH forms is associated with a pKa of 8.7 at 0 °. This equilibrium is perturbed by the binding of C2H2 in favor of the high pH form. Oxidation of Kpl, Cvl, and Cpl with the redox dye Lauth's violet bleaches 2a B. E. Smith, D. J. Lowe, and R. C. Bray, Biochem. J. 135, 331 (1973).

770

NITROGENFIXATION

[70]

4.32 3.63

1

2.009

FIG. 2. EPR spectrum of S2042--reduced Kpl at pH 6.8. At this pH only the low pH form is observed. The spectrum was recorded at 12°K with a microwave frequency of 9.16 GHz and a microwave power of 150 mW. Kpl was at 12.3 mg protein/ml. [M. J. O'Donnell, B. E. Smith, and D. J. Lowe, unpublished.]

the EPR signal without loss of activity. 12,26,27Oxidation with air, or excess Kz[Fe(CN)6] results in the loss of activity and the appearance of a complex series of signals near g = 2. During turnover, i.e., in the presence of Fe protein, MgATP and $2042-, the EPR spectrum of the S2042--reduced Mo-Fe protein is 90% bleached. Correlation with M6ssbauer data (see below) indicates that this is due to a further reduction, not oxidation, of this EPR center, lz'15 In the steady~state EPR-active intermediates present in low concentration with signals comparable to those observed in simpler Fe-S proteins have been reported (see Table IV). The intensities of these signals are perturbed by CzH2, C2H4, the product of C2H2 reduction, and the inhibitor CO. a°,al EPR signals which correspond to the -1 and - 3 oxidation of Fe4S4 clusters are observed (see Table IV). The signals observed under these conditions presumably arise from the redistribution of electrons within the redox centers of the Mo-Fe protein so that EPR active intermediates accumulate to a detectable level. They provide evidence for the binding of substrate product and inhibitor to the Mo-Fe protein, although a0 D. J. Lowe, R. R. Eady, and R. N. F. Thorneley, Biochem. J. 173, 277 (1978). a~ R. H. Burris and W. H. Orme-Johnson, Proc. Int. Symp. Nitrogen Fixation, 1st, 1974 Vol. 1, p. 248 (1976).

[70]

NITROGENASE

771

the lack of linewidth broadening observed with ~2C compounds a°'z2 does not support direct binding to the Fe4S4 clusters as occurs with hydrogenase .33 The M6ssbauer spectra of 5~Fe-enriched Kp134 and Av128 are very similar despite the difference in metal content of the proteins used in these studies. The low temperature spectra of the S204Z--reduced proteins are complex and consist of three overlapping quadrupole doublets and a component with paramagnetic hyperfine structure. At higher temperatures an additional quadrupole doublet appears, derived from the paramagnetic component at lower temperatures (Fig. 3). The spectral parameters indicate that Fe atoms are in three major environments. In both proteins approximately 55% of the Fe atoms are ferrous; 2 Fe atoms in Kpl and 3-4 Fe atoms in Avl have been assigned to spin-coupled highspin ferrous atoms. The remainder of the ferrous Fe is low spin: 8 Fe atoms in Kpl and 8-10 Fe atoms in Avl. The doublet which appears on raising the temperature has been correlated with EPR active center of the S2042--reduced proteins and is associated with 8 Fe atoms in Kpl and 8- i0 atoms in Av I. The M6ssbauer parameters of this species at 77 ° K (AE = 0.71, 8 -- 0.37) are similar to those of the Fe4S4 cluster of oxidized HiPiP. During turnover this is the only component of the spectrum which changes; and a new species with parameters close to those of reduced HiPiP (AE 1.12, 8 = 0.42) appears. These data allowed the assignment of the bleaching of the EPR signal of the S2042--reduced protein which occurs under these conditions to a "'superreduction," rather than an oxidation, a4 Kpl oxidized with the redox dye Lauth's violet retains full activity but the overlapping quadrupole doublets observed at 77°K in the S204Z--reduced protein are replaced by a single doublet (AE = 0.75, 8 -- 0.37). Mo-Fe proteins isolated from different sources show a considerable variation in redox potential and in the number of electrons involved in these processes. ~z,15 The midpoint potential of Cpl and Cvl has been measured by the EPR potentiometric technique. In this method s5 the potential is poised by the addition of $2042--or Ka[Fe(CN)6] to the protein plus redox mediator under anaerobic conditions and the system allowed to equilibrate for l0 min. The potential is then recorded and a sample removed for the measurement of the intensity of the g = 3,7 feature of the EPR spectrum. One electron az L. C. Davis, M. T. Henzl, R. H. Burris, and W. H. Orme-Johnson, Biochemistry (in press). aa D. L. Erbes, R. H. Burris, and W. H. Orme-Johnson, Proc. Natl. Acad. Sci. U.S.A. 72, 4792 (1975). a4 B. E. Smith and G. Lang, Biochem. J. 137, 169 (1974). a5 p. L. Dutton, Biochim. Biophys. Acta 226, 63 (1971).

772

NITROGEN FIXATION

:

/',.

[70]

,./

./W ~

-'..

.

/

.~...

•°

. ...

"~'~'~,.~

•

... - j

;-,

..",---,,,. " ...

.

..

.

..~."

.." , - ~ , ~ , - .~'4 2~K'~'---

•. . : . . . : ,

[ 2%

"-.vr'\ .,; ,.:-.; "

"._"

".."

I

I

I

-2

0

2

VELOCITY

RELATIVE

TO

Fe

4

M E T A L (mm/sec)

FIG. 3. M 6 s s b a u e r s p e c t r u m Of S2042--reduced Kpl enriched with STFe. (a) 4.2 ° K, zero magnetic field. (b) 4.2 ° K with a 100 G magnetic field parallel to the T-ray beam. (c) 4.2 ° K with a 550 G magnetic field perpendicular to the T-ray beam. [After B. E. Smith and G. Lang, Biochem. J. 137, 169 (1974).]

processes were observed for Cp 1 which gave a single Em,7.5 of - 20 m V , 36 and for C v l where two centers with Era,7.5 of - 6 0 mV and - 2 6 0 mV were observed. 37 At lower potentials, a 30% decrease in the intensity of the E P R signal of Cvl occurred with an Era,7.5 of - 4 6 0 mV. 3s. The value for Cp 1 is higher than that obtained by the dye-equilibration method where a four electron process was associated with an Em,7.5 of - 7 0 mW. 39 Electrolytic reduction of A v l oxidized with methylene blue gives two

36 W. G. Zumft, L. E. M o r t e n s o n , and G. Palmer, Eur. J. Biochem. 46, 525 (1974). 37 S. L. Albrecht and M. C. W. Evans, Biochern. Biophys. Res. Commun. 55, 1009 (1973). 3s M. C. W. E v a n s and S. L. Albrecht, Biochem. Biophys. Res. Commun. 61, 1187 (1974). 39 M. Walker and L. E. Mortenson, Biochem. Biophys. Res. Commun. 54, 669 (1973).

[70]

Nm~O~ENASE

773

TABLE V MOLECULAR WEIGHT AND SUBUNIT COMPOSITION OF FE PROTEINS Cp2 Gel filtration

Kp2

Av2

Ac2

64,000 a 66,500 e --

63,000 g

RI2

55,000 a 56,000 b Sedimentation -velocity A m i n o acid -composition Sum of subunit 55,000 a MW

62,000 c

69,200 c

66,000 a 68,000 e

61,000 u

64,000 h

Subunit one type composition Subunit MW 27,500 a'b

one type

one type

one type

one t ype

34,600 c

33,000 a~

30,800 ~

32,000 h

68,000 c 67,800 c

--

-67,000 °

65,000 h ---

a G. N a k o s and L. E. Mortenson, Biochemistry 10, 455 (1971). b M.-Y. W. Tso, Arch. Microbiol. 99, 71 (1974). c R. R. Eady, B. E. Smith, K. A. Cook, and J. R. Postgate, Biochern. J. 128, 655 (1972). d D. K l e i n e r and C. H. Chen, Arch. Microbiol. 98, 100 (1974). e W. A, Bulen, Proc. Int. Syrup. Nitrogen Fixation, 1st, 1974 Vol. 1, p. 177 (1976). r R. H. Swisher, M. Landt, and F. J. Reithel, Biochem. Biophys. Res. Cornmun. 66, 1476 (1975). g M. G. Yates and K. Planque, Eur. J. Biochem. 60, 467 (1975). h M. J. Whiting and M. J. Dilworth, Biochim. Biophys. Acta 371,337 (1974).

reduction w a v e s at - 3 2 0 and - 4 5 0 mV, both associated with a six electron reduction. 4°

Properties of the Fe Proteins The most useful criterion of purity for these proteins is electrophoresis in 8-10% polyacrylamide gels containing SDS. Interpretation of electrophoresis patterns obtained under nondenaturing conditions is complicated by the formation of size and charge isomers unless extreme precautions are taken to exclude oxygen. Oxygen damage results in a characteristic banded pattern on the gel and makes the identification of contaminating proteins difficult. The Fe proteins are unstable to prolonged storage except at liquid nitrogen temperatures. There is some variability in stability to storage at temperatures near 0° Kp2 and Ac2 are stable but some 40 G. D. Watt and W. A. Bulen, Proc. Int. Symp. Nitrogen Fixation, 1st 1974 Vol. 1, p. 248 (1976).

774

NITROGEN FIXATION

[70]

TABLE VI IRON AND SULFIDE CONTENT OF VARIOUS HIGHLY PURIFIED FE PROTEINS

Fe content (gm atom/mole) Ac2 RI2 Kp2 Cp2

4a 3.1 b 4c 4a 3-4 e

Specific activity S 2- content (nmole C2H2 reduced (gm atom/mole) mirdmg/protein) 3.9 -3.8 4 3-4

2000 434 980 2708 3100

MW 65,400 65,000 66,800 55,000

a M. G. Yates and K. Planque, Eur. J. Biochem. 60, 467 (1975). b M. J. Whiting and M. J. Dilworth, Biochim. Biophys. Acta 371, 337 (1974). c R. R. Eady, B. E. Smith, K. A. Cook, and J. R. Postgate, Biochem. J. 128,655 (1972). a G. Nakos and L. E. Mortenson, Biochemistry 10, 455 (1971). e M.-Y. W. Tso, Arch. Microbiol. 99, 71 (1974).

preparations of Av2 and Cp2 rapidly lose activity, x5 The extreme oxygen sensitivity of these proteins has been referred to in the context of the problems it poses to their purification; 45 sec is a typical half-life for the rate inactivation by air; oxygen damage is accompanied by the oxidation of sulfhydryl groups and loss of tertiary structure. ATP has been shown to enhance the sensitivity of Kp2 and Ac2 to oxygen. 41'42 The native molecular weights of highly purified Fe proteins from various organisms are shown in Table V. Cp2, with a MW of 55,000, is smaller than other Fe proteins investigated, which have average molecular weights in the range 62,000-66,000. The subunit structures of these proteins have been investigated by SDS electrophoresis, and in all cases a single band was observed. Ultracentrifuge data for Av2 in 8 M urea are consistent with a single species of MW 30,000. These data are summarized in Table V and are consistent with the Fe proteins being dimers comprised of identical subunits. In the case of Cp2, this assignment is supported by amino acid sequence studies. 43 The amino acid compositions of Cp2, Ac2, Kp2, and RI2 are similarlS; tryptophan is absent, and, as with the Mo-Fe proteins, acidic residues 41 R. R. Eady, B. E. Smith, R. N. F. Thorneley, M. G. Yates, and J. R. Postgate, in -Nitrogen Fixation by Free-living Microorganisms" (W. P. D. Stewart, ed.), p. 377. Cambridge Univ. Press, London and New York, 1975. 42 M. G. Yates, FEBS Lett. 8, 281 (1970). 43 M. Tanaka, M. Haniu, K. T. Yasunobu, and L. E. Mortenson, in - I r o n and Copper Proteins" (K. T. Yasunobu, H. F. Mower, and O. Hayaishi, eds.), p. 83, Plenum, New York, 1976.

[70]

NITROGENASE

775

(about 20%) are about twice as abundant as basic residues. The amino acid sequence of Cp2 has been determined. Unlike the ferredoxins, the 6 cysteine residues that each subunit of this protein contains are not clustered but are widely distributed among the 273 residues of the polypeptide chain. 43 The Fe proteins contain approximately 4 gm atoms each of Fe and Sz- per molecule (see Table VI). Cluster extrusion techniques using thiolate ligands have recently been used to establish that these are present in a single Fe4S4 cluster in Cp244"45 (this volume, [71]). How the single Fe4S4 cluster is distributed between the two identical subunits is not known, but from the distribution of cysteine residues in the protein it has been suggested that it bridges the two subunits. At temperatures below 30°K S2042--reduced Fe proteins have a rhombic EPR spectrum with gav = 1.94 similar to those of reduced ferredoxins (see Table VII). The intensity of this signal depends on the source the protein was isolated from, but always integrates to less than 1 electron/ mole. Oxidation of Cp227 and Ac22 with a tenfold excess of PMS bleaches the EPR signal without significant loss of activity. Stopped-flow studies have shown that on rereduction of oxidized Ac2 with SOz-" (derived from the dissociation of $2042-), 1 electron is rapidly (K > 108 M -1 sec -a) taken up and the EPR signal fully restored. 4n Spectral measurements indicate that a second electron is subsequently taken up in three slow phases without further change in the EPR spectrum. This behavior is difficult to reconcile with a redox process involving a single Fe4S4 cluster that this protein is thought to contain. During turnover the EPR signal is 90% bleached indicating that the protein is predominantly in the oxidized form. The M6ssbauer spectrum of ~Fe substituted S2042--reduced Kp234 at 195°K is a symmetrical doublet (8 0.52 mm/sec, AE 1.05 mm/sec) which at 4.2°K broadens into a multiplet indicating magnetic character (Fig. 4). At 77 ° K a minor species at +1.3 mm/sec is apparent in the spectrum, indicating some differences in the environment of the Fe atoms. These spectra are very similar to those of reduced ferredoxins which contain Fe4S4 clusters 47 and are strikingly different from reduced ferredoxins 44 W. H. Orme-Johnson, L. C. Davis, M. T. Henzl, B. A. Averill, N. R. Orme-Johnson, E. Munck, and R. Zimmerman, in -Recent Developments in Nitrogen Fixation" (W. Newton, J. R. Postgate, and C. Rodriguez Barrueco, eds.), p. 131. Academic Press, New York, 1977. 4s W. O. Gillum, L. E. Mortenson, J.-S. Chen, and R. H. Holm, J. A m . Chem. Soc. 99, 584 (1977). 46 M. G. Yates, R. N. F. Thorneley, and D. J. Lowe, FEBS Lett. 60, 89 (1975). 47 R. N. Mullinger, R. Cammack, K. K. Rao, D. O. Hall, D. P. E. Dickson, C. E. Johnson, J. D. Rush, and A. Simopulos, Biochem. J. 151, 75 (1975).

776

NITROGENFIXATION

[70]

T A B L E VII E P R SIGNALS OF HIGHLY PURIFIED FE PROTEINS

(spin/mole)

Specific activity (nmole C 2 H 2 reduced min/mg protein)

0.45 - 0.07 0.17b-0.24 c -0.25 0.79 0.2

600-1500 a 2000 b 1815 d 2400 f 1200 e 2200 u

Integrated intensity g Values Kp2 Ac2 Av2 Cp2

2.053 2.05 2.05 2.06 2.06 2.05

1.942 1.94 1.94 1.94 1.94 1.94

1.865 1.87 1.88 1.88 1.88 1.87

a B. E. Smith, D. J. L o w e , and R. C. Bray, Biochem. J. 135, 331 (1973). M. G. Yates and D. J. L o w e , Eur. J. Biochem. 60, 467 (1975). c M. G. Yates, R. N. F. Thorneley, and D. J. L o w e , FEBS Lett. 60, 89 (1975). d V. K. Shah and W. J. Brill, Biochirn. Biophys. Acta 305, 445 (1973). e W. H. O r m e - J o h n s o n , W. D. Hamilton, T. Ljones, M.-Y. W. Tso, R. H, Burris, V. K. Shah, and W. J. Brill, Proc. Natl. Acad. Sci. U.S.A. 69, 3142 (1972). s W. H. O r m e - J o h n s o n , L. C. Davis, M. T. Henzl, B. A. Averill, N. R. Orme-Johnson, E. Munck, and R. Z i m m e r m a n , in "'Recent D e v e l o p m e n t s in Nitrogen Fixation" (W. Newton, J. R. Postgate, and C. Rodriguez Barrueco, eds.), p. 131. A c a d e m i c Press, New York, 1977. g G. Palmer, J. S. Multani, Wo C. Cretney, W. G. Zumfl, and L. E. M o r t e n s o n , Arch. Biochem. Biophys. 153, 325 (1972).

containing Fe2S2 clusters, 4s where the two types of Fe atoms can be distinguished in the M6ssbauer spectra. The optical spectra of Kp2, Cp2, Ac2, and Rj2 consist of a broad absorption devoid of any major features, is The increase in absorption near 425 nm on oxidation has been used in presteady state studies on the electron transfer between the Fe protein and Mo-Fe protein 4~ and the reduction of oxidized Fe protein by SOz- .46,50The increase in absorbance of Cp2 on oxidation by Cp 1 plus ATP in the presence of limiting Na2S204 parallels the decrease in intensity of the EPR signal of Cp2. 51 The circular dichroism spectra of Kp2 and Cp2 do not show any transitions in the visible region, la'2° in contrast to the ferredoxins. The a-helix content of both proteins decreases markedly on 02 inactivation. The redox potential of Cp2 has been determined using both optical and 4s W. R. D u n h a m , A. J. Bearden, I. T. Salmeen, G. Palmer, R. H. Sands, W. H. OrmeJ o h n s o n , and H. Beinert, Biochim. Biophys. Acta 253, 134 (1971). 4a R. N. F. Thorneley, Biochem. J. 145, 391 (1975). 50 R. N. F. Thorneley, M. G. Yates, and D. J. L o w e , Biochem. J. 155, 137 (1976). 5~ T. Ljones, Biochim. Biophys. Acta 321, 103 (1973).

[70]

NITROGENASE

777 w

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.

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:

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.

•

-.

:,-s"." ' "."-".::." "•

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/

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77°K

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195°K

..::.

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a

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I

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2

VELOCITY

RELATIVE

TO

Fe

METAt(mm/sec)

FIG. 4. M 6 s s b a u e r s p e c t r u m o f S 2 0 4 ~ - - r e d u c e d Kp2 e n r i c h e d w i t h 5rFe. (a) 4.2 ° K with a 100 G m a g n e t i c field parallel to the y - r a y b e a m . (b) 4.2°K w i t h a 550 G m a g n e t i c field p e r p e n d i c u l a r to the y - r a y b e a m . [After B. E. S m i t h and G. L a n g , B i o c h e m . J. 137, 169 (1974). ]

EPR potentiometric techniques. In the optical method the S2042--reduced protein is titrated with various oxidized redox dyes, and after equilibration the potential determined by spectral measurements of the dye which can be correlated with the change in protein absorbance. This technique gives a midpoint potential Em,r.5 = - 2 4 0 mV, and an extrapolated value of 1.4-2 electrons to reduce fully oxidized Cp2. 39 The number of electrons required for this process is in good agreement with the value of 2 obtained 46 for the reduction of PMS-oxidized Ac2 by $2042-. The redox properties of Cp2 have been investigated using the EPR potentiometric technique and the H+/H2 hydrogenase couple 31 or SzO4~- and K3Fe(CN)6 to poise the potential.Sn From the variation in intensity of the EPR signal, values of Em,7.s of -294 --- 20 mV and -270 mV were obtained. As discussed below, a decrease in potential to - 4 0 0 or -490 mV was ob-

778

NITROGENFIXATION

[70]

served 31"aein the presence of MgATP and to -380 mV in the presence of MgADP. Binding of ATP to Cp2, Kp2, and Ac2 has been demonstrated by gel filtration in columns equilibrated with 14C-labeled nucleotide. 15 More recently 52 gel equilibration studies have given quantitative data for Cp2. MgATP binds to two sites with a K0 = 16.7/zM; MgADP inhibits the binding of MgATP to one of these sites and increases the affinity of the uninhibited site for MgATP approximately twofold. MgADP binds with a Kd = 5.2 /~M. The MgATP-induced change in the EPR signal from rhombic to axial symmetry has also been used to monitor the binding of MgATP to Cp2 and Kp2. The data are consistent with tight binding of 2 molecules of ATP to Cp2 but looser binding to Kp2 with Ko = 0.4 mM. Problems associated with the interpretation of binding as monitored by EPR changes are discussed in Orme-Johnson and Davis 13 and Eady and Smith. ~5 The change in conformation caused by MgATP binding is reflected in a large increase in titer and reactivity of sulphydryl groups in Kp2 to DTNB 53 and Cp2 by the increased reactivity of the Fe atoms with a , a ' - b i p y r i d y l . 54 MgADP does not alter the latter rate but increases sulfhydryl group reactivity, although to a lesser extent than MgATP. The midpoint potential of Cp2 ( - 294 +-- 20 mV) is decreased to -4 0 0 ___ 20 mV by binding of MgATP. MgADP has a similar effect. The changes in conformation which accompany the decrease in redox potential are the consequence of binding not hydrolysis of ATP. ~1 The 110 mV decrease in potential has been attributed 3e to the 70-fold tighter binding of MgATP to the oxidized Fe protein with Kd = 0.3 /~M. 52M.-Y. W. Tso and R. H. Burris, Biochim. Biophys. Acta 309, 263 (1973). R. N. F. Thorneleyand R. R. Eady, Biochem. J. 133, 405 (1974). 54G. A. Walker and L. E. Mortenson,Biochern. Biophys. Res. Commun. 53, 904 (1973).