E @ * # Acta Botanica Sinica 2003,45 (7): 820-824

Crystallization of Nitrogenase MoFe Protein (NifB-Avl) from a nifB Mutated Strain U W45 of Azotobacter vinelandii

ZHAO Yingl, LU Yu-Bingl, ZHAO Jian-Fengl, ZHOU Jun-Xian2, QlAN Zhong-Xing1,

WANG Yao-Ping2', HUANG Ju-Ful'

(1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, lnstitute of Botany, The Chinese Academy of Sciences, Beijing 100093, China;

2. Institute of Biophysics, The Chinese Academy of Sciences, Beijing 1001 01, China)

Abstract: Six hundred and 28 mg of NifB- Avl was obtained by a chromatography twice on DE 52 columns and Sephacryl S-300 column from the crude extract (37 677 mg) of a nifS mutated strain UW45 of Azotobacter vinelandii Lipmann. The protein was almost homogeneous as determined by Cwmassie staining of SDS gels. The analysis by SDS-PAGE showed that NifB-Avl was similar to Avl from wild-type strain of A. vinelandii (OP) in the kinds of subunits ( a and B subunit). When complemented with Av2, NitE-Avl had hardly any H-reducing activity, but could be significantly activated by FeMoco extracted from Avl. Under a suitable condition for crystallization, short dark-brown rhombohedra1 crystals could be obtained from NifB-Avl . Both of the longest sides of the biggest crystal were 0.1 mm. The time of the formation of crystals and number, size, quality and shape of crystals obviously depended not only on the kinds and concentrations of the components in the precipitant solution, but also on the methods for crystallization and technical bias, etc. The preliminary results showed that the crystal seemed to be fomed from NifB-Avl . Key words: mutant strain UW45 of Azotobacter vinelandii; nitrogenase NifB-Avl; crystallization

Mo-containing nitrogenase is composed of two sepa- rable proteins called MoFe protein (Avl) and Fe protein (Av2). FeMoco and Pcluster in Avl play a very important role in the structure and function of the protein (Kim and Rees, 1992). The biosynthesis and insertion of FeMoco in nitrogen fixation bacteria in vivo are controlled by nifB, nifN, nifE, nifQ, nifb', nifH and nifX (Allen et a/, 1995; Rangaraj et al, 2001), and the biosynthesis of P-cluster is controlled by at least nEfZ (Huang etal, 1999). Therefore, the structure and function of these nitrogenases from these genes deleted or mutated strains have been vigorously studying. Since 1977, a crudeextract from UW45, which is a nifS point-mutation strain, has been used as an Apo-Avl activated by FeMoco and its chemical model in order to study the structure and function of both nitrogenase and the model(Shah and Brill, 1977). And the protein was puri- fied to >95% purity in 1990 (Paustian etal, 1990). Since UW45 deletes a NifB-cofactor (iron- and sulfur-contain- ing precursor to FeMoco), it only synthesizes a FeMoco-deficient NifB-Avl which is similartoAv1 in the structure of Pclusters and the environment of polypep- tides around the clusters (Allen etal, 1995; Christiansen et al, 1998). Dislike AnitHAvl from nwdeleted strain DJ54 (Tal etal, 1991), the insertion of FeMoco into NifB-Avl

in vitro is independent on an incubation with both Av2 and MgATP. Acomparative study on their exact structures should help us to understand well the mechanism of recon- stitution in vitro of the protein with FeMoco and its chemi- cal model. One of the biggest and most important break- throughsofstrqctural studieson nitrogenase has been made after the completion of an X-ray diffraction analysis of crys- talsofAv1 and its complex proteinswithAvZ!(Kim and Rees, 1992; Schindelin etal, 1997). It is reasonable to think that the X-ray diffraction analysis of crystals of clusterdeficient Avl from mutant strains is also of great importance forfur- ther understanding the mechanism of nitrogen fixation. However, the growth of crystal suitable for the X-ray dif- fraction analysis is usually a main hindrance for crystallog- raphy(McPherson ,1983). Therefore, itisnecessarytoscreen the optimal conditions for crystallization and crystal growth of NifB-Avl .

I Materials and Methods Growth of mutant strain UW45ofAzotobactervinelandii

Lipmann and purification of NifB-Avl and NifB-Av2 from the strain were carried out according to Shah and Brill (1 977) and the modified method of Paustian et al(l990), respectively.

Received: 2002-05-08 Accepted: 2002-08-28 Supported by the State Key Basic Research and Development Plan of China (001CB1089-06), the National Natural Science Foundation of China (30270296) and the National Manned Space Engineering Project of China. *Author for correspondence.

ZHAO Ying et al: Crystallization of Nitrogenase MoFe Protein (NifB-Av1) from a nifB Mutated Strain UW45 of Azotobacter vinelandii 821

In order to prevent the NifB-Av1 from a damage re-sulted possibly from heat treatment at 52 ℃, the crude ex-tract of UW45 was not heated. The crude extract (37 677mg) was applied to DE52 column, followed by developingwith NaCl gradient (0.1-0.5 mol/L) in 25 mmol/L Tris buffer(pH 7.4) containing 0.3 mg/mL Na2S2O4 (DT). After chro-matography again on the second DE52 columns, the par-tially purified NifB-Av1 (1 922 mg) and NifB-Av2 (1 017 mg)were obtained. The NifB-Av1 was further purified onSephacryl S-300 column (2.5 cm× 72.0 cm) and concen-trated on a small DE52 column.

Extraction of FeMoco from Av1 were performed accord-ing to the methods of Shah et al (1997) and Huang et al(1999).

Determination of protein concentration and substrate-reducing activity, and SDS-PAGE were carried out accord-ing to Shah and Brill (1977) and Paustian et al (1990).

Crystallization of NifB- Av1 was performed according toDrenth et al (1991) and Huang et al (1999). Unless de-scribed elsewhere, the crystals were grown by vapor diffu-sion using the hanging drop method for 7 d at 20 ℃.

All of the operations were done under Ar. All of thesolutions used were rigorously degassed and filled withAr. The concentrations of DT, Tris, and NaCl in the NifB-

Av1 solution were 0.3 g/L, 25 mmol/L and 250 mmol/L. Theconcentration of DT in precipitant solution was 12.0mmol/L.

2 Results and Discussion2.1 Identification of NifB-Av1

The NifB-Av1 (628 mg) from the last DE52 column wasbasically homogeneous as determined by Coomassie stain-ing of SDS gels, and both the positions and amount ratio oftwo main bands were almost the same as those of αand βsubunits from Av1 (Fig. 1). And a mixture of NifB- Av1 withAv1 on the gel showed that the bands for the two proteins

well overlapped each other, respectively. This indicatesthat kind and composition of the NifB- Av1 are almost thesame as those of Av1.

As reported previously in other laboratories (Shah andBrill, 1977; Christiansen et al, 1998; Huang et al, 1999), thecrude extract of UW45 hardly exhibited any C2H2-reducingactivity, but was able to be efficiently complemented withAv1 (Table 1); and either the crude extract or NifB- Av1could be significantly reactivated by FeMoco extracted fromAv1 with NMF (Table 2). The activities of the Av1 comple-mented with the partially purified NifB- Av2 were almost thesame as that of the same batch Av1 complemented withAv2 (Huang et al, 1999). This indicates that NifB- Av2 issimilar to Av2 in the activity and FeMoco can activate theinactive NifB-Av1 in vitro .2.2 Crystallization of NifB-

Av1Besides the property, purity and concentration of a

protein, there are many factors affecting crystallization ofthe protein, such as the kind, concentration and ratios of allcomponents in the precipitant solution, method ofcrystallization, pH of the solution, temperature and techni-cal bias (Schindelin et al, 1997; McPherson, 1983; Drenthet al, 1991; Kim and Rees, 1992; Huang et al , 1999).Therefore, it is necessary to optimize the above conditionsin order to obtain crystals of NifB- Av1 suitable for X-ray

Fig.1. SDS-PAGE (6.0%) of NifB-Av1. 1 and 5, NifB-Av1(5.1 and 2.5 µg); 2 and 4, mixture of NifB-Av1 (2.5 µg) with Av1(2.5 and 7.5 µg); 6 and 8, mixture of NifB-Av1 (2.5 µg) with Av1(2.5 and 5.0 µg); 3 and 7, Av1 (7.5 and 5.0 µg).

Table 1 Complement of Av1 with the NifB- crude extract andpartially purified NifB- Av2

SampleTotal activity Specific activity of Av1(4)

(nmol C2H

4/min) (nmol C

2 H

4.min-1.mg-1 pro.)

NifB- CE(1) 0 -NifB- CE(1) + Av1(2) 92.40 1 224NifB- Av2(3)+ Av1 (2) 169.14 2 192(1), UW45 crude extract (4.30 mg); (2), Av1 (0.075 mg); (3), par-tially purified NifB- Av2 (0.62 mg); (4), specific activity of Av1calculated after subtraction of activity induced by NifB- Av2 alone.

Table 2 Activation of NifB- Av1 by FeMocoSample Total activity Specific activity (nmol H

2/min) NifB- Av1 FeMoco

(nmol H2.min-1.mg-1) (nmol H2

.min-1.nmol-1)

NifB- CE (1) 0 0 -NifB- CE (1)+ 4.10 1 12FeMoco (2)

FeMoco (2)+ 35.57 (4) 847 (4) 310 (4)

NifB- Av1 (3)

(1), UW45 crude extract (4.300 mg); (2), NifB- CE (4.300 mg) wasincubated with FeMoco extract (0.340 ng Mo atoms) from Av1 for70 min at 30 ℃; (3), NifB-Av1 (0.042 mg) was incubated withFeMoco extract (0.115 ng Mo atoms) from Av1 for 70 min at 30℃; (4), complemented with NifB- Av2 (0.620 mg), the activity wascalculated after subtraction of activity induced by NifB- Av2 incu-bated with NMF.

822 植物学报 Acta Boanica Sinica Vol.45 No.7 2003

diffraction analysis.2.2.1 Effect of precipitant solution on the crystallization

Hepes buffer Within the Hepes concentrations shownin Tables 3 and 4 (pH 8.2), the possibility of nucleation ofNifB- Av1 in the first week increased obviously with theincrease of Hepes concentration. In the precipitant solu-tion #1 containing 51 mmol/L Hepes shown in Table 3, acrystal formed in the second week could gradually grow.After incubation for four months, the longest two sizes anddiagonals of the crystal were 0.10 mm × 0.10 mm and 0.17mm×0.09 mm, respectively (Fig.2). It is indicated that theconcentration of the buffer in the precipitant solution couldaffect the electric charge and conformation of protein, re-sulting in a change of the possibility and rate of nucleation.

PEG 8000 Under the conditions shown in Table 4, theconcentration of PEG 8000 had also a significant effect onthe crystal number and size of NifB- Av1. In comparisonwith ΔnifZ Av1 (Huang et al, 1999), the optimal PEG con-centration for crystallization of NifB- Av1 at which the crys-tals were bigger in size and fewer in number was slightlyhigher. The protein crystals could not be formed or a largeamount of small crystals were formed when the PEG con-centration was out of this range.

PEG could absorb water from the environment aroundthe protein molecules, resulting in decreasing protein dis-solution (Drenth et al, 1991). When the PEG concentrationwas lower than a critical value, its crystals could not be

formed. When the concentration was too high, the accel-eration or disturbance of crystal formation was resultedfrom the acceleration of loss of water from the protein, lead-ing to the following possibilities that the crystalline nucleicould not be formed or were too much. Only under theoptimal concentration of PEG, the rate of losing water couldbenefit a formation of a few nuclei and their slow growth,leading to formation of the single and big crystals of goodquality.

MgCl2 and NaCl The concentration of MgCl2 couldsignificantly affect the number and size of crystals from themutant protein (Table 5). In comparison with ΔnifZ Av1(Huang et al, 1999) , the optimal concentration (about 250

Table 3 Effect of Hepes concentration on crystallization of NifB- Av1 by vapor diffusion method in the hanging drop

Solution(1)

Hepes concentration (mmol/L) 51 61 71 81

Crystal number Crystal size Crystal number Crystal size Crystal number Crystal size Crystal number Crystal size #1 0 - 0 - 0 - 25 Small #2 0 - 1 Small(2) 0 - 5 Small #3 0 - 2 Small(2) 13 Small 8 Middle #4 0 - 0 - 2 Small 3 Small(1), the concentrations of NifB-Av1 and MgCl

2 were 8.48 mg/mL and 598.95 mmol/L, respectively, and the concentrations of PEG 8000 and

NaCl in solution #1, #2, #3 and #4 were 5.50% and 740.14 mmol/L, 5.00% and 740.14 mmol/L, 4.75% and 490.14 mmol/L, and 4.30% and490.14 mmol/L, respectively; (2), crystal of bad quality (sides of crystals were not sharp).

Table 4 Effect of the concentration of PEG 8000 on crystallization of NifB- Av1 by vapor diffusion method in the hanging drop

PEG %(1) Exp.#1 Exp.#2

Crystal number Crystal size Crystal number Crystal size 3.78 0 - 0 - 4.00 0 - 4 Small 4.22 0 - 1 Small 4.44 1 Middle 5 Middle 4.76 12 Small 5 Small 5.00 5 Small 5 Small 5.50 30 Small 20 Small(1), concentrations of Hepes in Exp.#1 and #2 are 61.04 and 81.36 mmol/L (pH 8.2), respectively; and concentrations of NifB- Av1, NaCland MgCl

2 in Exp. #1 and Exp. #2 are 8.48 mg/mL, 491.08 and 598.95 mmol/L, respectively.

Fig.2. Crystal of NifB- Av1 observed in a hanging drop twoweeks later (× 250). The protein and crystalline condition arethe same as those of crystalline solution #1 containing 51mmol/L Hepes in Table 3.

ZHAO Ying et al: Crystallization of Nitrogenase MoFe Protein (NifB-Av1) from a nifB Mutated Strain UW45 of Azotobacter vinelandii 823

mmol/L) for its crystallization was slightly lower. Like PEG,both MgCl2 and NaCl could not only absorb water mol-ecules from the environment around the protein, but alsorelease cations (Mg2+ and Na+, respectively), leading to aneffect of the crystalline process by a change of the electriccharges on proteins. In general, the effect increases withthe increase of the ion concentration and square chargenumber of cations (McPherson, 1983; Tal et al, 1991; Huanget al, 1999). A body of experiments in our laboratory exhib-ited that the effect of MgCl2 was obviously higher thanthat of NaCl.2.2.2 Effect of technical bias on the crystallization Un-der the same conditions for crystallization by the vapordiffusion using the hanging drop method, the crystalsformed in two drops sited on one slide were obviouslydifferent from each other in both the number and size, al-though their crystal qualities were not easy to be differen-tiated (Table 5). Two technical biases exhibited: (1) theerror of the very small sample volume which led to a sig-nificant change in the concentration; and (2) it was pos-sible to let the protein be differently mixed with the pre-cipitant solution when the slide was turned over and putagainst the reservoir. Therefore, it could make the watermolecules lose with different rates from the environmentaround protein molecules, leading to a different process of

crystallization.2.2.3 Effect of crystallization method on the crystalliza-tion Under the same conditions including the same pro-tein and precipitant solutions, the results obtained by thevapor diffusion was different from those by the liquid/liquid diffusion, and sometimes the differences were veryobvious. In one week, the crystals were not formed by thevapor diffusion, but more than 10 middle crystals wereobserved by the liquid/liquid diffusion using the same pro-tein and precipitant solution #4 containing 51 mmol/LHepes shown in Table 3 (Fig.3). In comparison with thecrystals shown in Fig.2, the crystals exhibited thicker.Both the rate and route for diffusion of water moleculesfrom the environment around protein molecules could bedifferent when the protein was crystallized by the differentdiffusion method, resulting in different processes forcrystallization.

The observation by the optical differential microscopeshowed that the crystals formed from the solution contain-ing NifB-Av1 could be crystals formed from a protein, notfrom compounds in the precipitant solution. The dark browncolor of crystals from both Av1 and ΔnifZ Av1 were relatedto the large amount of iron in the proteins (Huang et al,1999). Since NifB- Av1 still has P-cluster of which iron con-tent is about a half of Av1, the color of the crystal is reason-able to be brown. Since the NifB- Av1 used for crystalliza-tion was basically homogeneous judged by SDS-PAGE, itwas almost impossible that the crystals were from othercontamination proteins. Finally, like ΔnifZ Av1 (Huang etal, 1999), NifB- Av1 could be crystallized only under ananaerobic condition and the formed crystals disappearedafter an exposure to air. Besides the protein, only DT canbe oxidized in the crystalline system while DT crystal cannot be brown in color. Therefore, it seems to be reasonableto suppose that the formed crystals were those of theprotein. However, the final conclusion could be made onlyafter X-ray diffraction analysis of the crystals. The researchwork still goes on the way.Acknowledgements: The authors wish to thank Mr.ZHANG Hua-Feng for his help in some experiments.

Table 5 Effect of MgCl2 concentration on crystallization of NifB- Av1 by vapor diffusion method in the hanging drop MgCl

2(1) Drop #1(2) Drop #2(2)

(mmol/L) Crystal number Crystal size Crystal number Crystal size 276.00 10 Middle 3 Small 398.70 20 Middle 20 Small 499.12 10 Middle 20 Small 598.95 3 Small 1 Small 698.78 0 - 3 Small(1), concentrations of protein, PEG 8000, Hepes and NaCl were 8.48 mg/mL, 4.30%, 60.14 and 245.07 mmol/L, respectively; (2), two dropscontaining the same solutions on one slide.

Fig. 3 Crystals of NifB- Av1 observed in a small glass tube oneweek later (× 150). Fifteen µL of the solution #4 containing 51mmol/L Hepes and 15 µL of the protein shown in Table 3 wereadded to a glass tube (internal diameter was 0.1-0.2 mm).

824 植物学报 Acta Boanica Sinica Vol.45 No.7 2003

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棕色固氮菌突变种 UW45 的固氮酶钼铁蛋白(NifB-Av1)结晶

赵 颖 1 吕玉兵 1 赵剑峰 1 周军贤 2 潜忠兴 1 王耀萍 2* 黄巨富 1*

(1.中国科学院植物研究所光合作用与环境分子生理学重点实验室，北京 100093；

2.中国科学院生物物理研究所，北京 100101)

摘要： 经两次DE52和Sephacryl S-300柱层析，从棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW45粗

提液(37 677 mg蛋白)中纯化得到628 mg的NifB-Av1。经考马斯亮蓝R-250染色的SDS凝胶电泳分析表明，该蛋

白基本达到SDS凝胶电泳纯，组成它的亚单位的种类与Av1(α和β亚单位)相似。NifB-Av1不能与NifB-Av2重组成具

放氢活性的固氮酶，但可使与其保温重组的FeMoco显出高活性。在合适条件下，NifB-Av1可在结晶溶液中析出棕色短

斜四棱柱晶体，目前所得最大晶体的二维边长均为0.1 mm。能否出现晶体以及出晶时间、晶体数目、大小、质量和形

状等，与沉淀剂溶液各组分的种类和浓度、结晶方法、实验操作等因素密切相关。初步结果表明，所得晶体为NifB-Av1

单晶。

关键词: 棕色固氮菌突变种UW45；固氮酶NifB-Av1；结晶

中图分类号: Q945.13 文献标识码: A 文章编号: 0577-7496(2003)07-0820-05

收稿日期:2002-05-08 接收日期:2002-08-28基金项目:国家基础研究发展规划项目(项目 00 1CB108 9-0 6); 国家自然科学基金（29771033）; 国家载人航天工程项目。

* 通讯作者。

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(Managing editor: HE Ping)