全 文 ：Received 15 Jan. 2003 Accepted 23 Apr. 2003
Supported by the National Natural Science Foundation of China (30270296), the National Fundamental Research Project (“973” Program
001CB1089-06) and the National Manned Space Engineering Project of China (921-2).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Crystallization of MoFe Protein (DnifH Av1) from a nifH Deleted
Strain of Azotobacter vinelandii
BIAN Shao-Min1, ZHAO Jian-Feng1, LÜ Yu-Bing1, ZHAO Ying1, ZHOU Hui-Na1,
WANG Yao-Ping2, HUANG Ju-Fu1*
(1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China;
2. Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China)
Abstract: A FeMoco-deficient DnifH Av1 was partially purified from a nifH deleted mutant DJ54 of
Azotobacter vinelandii Lipmann grown in NH3-limited medium. By using the same purification method, DnifE
Av1 and NifB-Av1 were obtained from DJ35 and UW45, respectively. The latter two proteins were obviously
much purer than DnifH Av1. Under a suitable condition for crystallization, dark brown short rhombohedron
crystals could be obtained from the three proteins. Like NifB-Av1, the time for formation of DnifH Av1
crystal was longer than that of DnifE Av1. But the optimal concentrations of precipitant and buffer for
crystallization of DnifH Av1 were similar to those of DnifE Av1. SDS-PAGE analysis showed that the
crystalline DnifH Av1 was similar in the composition to OP Av1. It indicates that the crystal formed in DnifH
Av1 solution could be the protein crystal.
Key words: mutant DJ54 of Azotobacter vinelandii ; nitrogenase DnifH Av1; crystallization
The metalloenzyme nitrogenase complex from Azoto-
bacter vinelandii is composed of Av1 and Av2. Av2 is a
dimer of identical subunits encoded by the nifH gene. Av1
is a a2 b2 tetramer with a and b subunits encoded by the
nifD and nifK genes, respectively. Av1 contains two (8Fe-
7S) clusters (P-cluster) bridged between each a b subunits
pair and the (MoFe7S9 homocitrate) cluster (FeMoco) lo-
cated within each α subunits (Ribbe et al., 2002). FeMoco
synthesis requires seven nif gene products, NifN, NifE,
NifQ, NifX, NifV, NifB and NifH. The NifB produces an
iron- and sulfur-containing FeMoco precursor, presumably
the starting point of the FeMoco synthesis (Allen et al.,
1995). The NifH has three different roles in the nitrogenase
enzyme system. Apart from serving as the physiological
electron donor to Av1, NifH is involved in FeMoco biosyn-
thesis and in maturation of the FeMoco-deficient Av1
(Rangaraj et al., 1999). FeMoco-deficient, but P-cluster con-
taining Av1 have been proven to be useful for the two
major aspects of nitrogenase research, the maturation of
Av1 and the feature of the P-cluster. Two types of 100%
FeMoco-deficient Av1 have been isolated and
characterized, DnifH Av1 and DnifB Av1 are presumably
different catalytically and structurally (Tal et al., 1991;
Gavini et al., 1994; Ribbe et al., 2002). The exact function
and redox properties of the P-cluster have been the focus
of discussion for a long time (Burgess and Lowe, 1996). A
comparative study of the DnifH Av1 and DnifB Av1 can be
used to address this problem as well as many other remain-
ing questions about the function and assembly of the P-
cluster (Ribbe et al., 2002). The crystal structure of DnifB
Av1 from DJ1143 has been reported (Schmid et al., 2002).
Recently, DnifE Av1 was purified and crystallized (Zhao et
al., 2003a; 2003b). Hence, a comparative study on crystal
structures of DnifH Av1 with DnifB Av1 and DnifE Av1 is of
importance. This present study was undertaken for the at-
tempt to select the optimum conditions for crystallization
of DnifH Av1.
1 Materials and Methods
nifH-deletion mutant strain DJ54 of Azotobacter
vinelandii Lipmann was kindly provided by Prof. Burgess
in Department of Molecular Biology and Biochemistry.
Purification, determination of protein concentration and
SDS-PAGE analysis of DnifH Av1, DnifE Av1 and NifB-
Av1 were carried out by using the methods of Gavini et al.
(1994) and Zhao et al. (2003a; 2003b).
Crystallization of the three apo-Av1 by vapor diffusion
in the hanging drop method and the liquid/liquid diffusion
method was carried out according to the method of
Mcpherson et al. (1983), Huang et al. (2000) and Zhao et
Acta Botanica Sinica
植 物 学 报 2004, 46 (1): 58-62
BIAN Shao-Min et al.: Crystallization of MoFe Protein (DnifH Av1) from a nifH Deleted Strain of Azotobacter vinelandii 59
al. (2003), respectively.
Several crystals of DnifH Av1 were picked and dissolved
with the 0.05 mL 0.30 mmol/L NaCl in Tris buffer, then vi-
brated and centrifuged for 3 min at 12 000 r/min. The super-
natant was called crystalline DnifH Av1.
2 Results
2.1 The comparison in crystallization of DnifH Av1 with
DnifE Av1 and NifB-Av1
After chromatography twice on DE52, then by further
purified on Sephacryl-300 and Q-Sepharose columns, DnifH
Av1 and DnifE Av1 and NifB-Av1 were obtained,
respectively. Both DnifE Av1 and NifB-Av1 were much
purer than DnifH Av1 (Fig.1). Gavini et al. (1994) reported
that after chromatography on DE52, Q-Sepharose columns
and ACA34 columns, DnifH Av1 could be purified to be
indistinguishable from crystalline OP Av1 by the criteria of
SDS- PAGE, but before the chromatography on the ACA34
column three times, the protein was similar in the purity to
DnifH Av1 in Fig.1A. It is shown that the protein in Fig.1A
was normal.
In 14 optimal solutions including nine solutions in Fig.1
for crystallization of DnifZ Av1, nitrogenase MnFe protein
and CrFe protein (Huang et al., 2000; Lü et al., 2003), both
DnifH Av1 and NifB-Av1 were crystallized in only one so-
lution and DnifE Av1 was crystallized in six solutions at the
first 9 d. But after a further incubation, the former two pro-
teins could also be crystallized in the other four solutions.
Like DnifE Av1, the crystals of DnifH Av1 in the most solu-
tions were bigger than those of NifB-Av1 (Table 1). The
analysis of the crystalline DnifH Av1 by SDS-PAGE showed
that the protein was similar in the purity to OP Av1 and was
much purer than DnifH Av1 before crystallization (Fig.1B).
This indicates that the crystalline protein seems to be the
DnifH Av1. Under the same condition, a precipitated pro-
tein in the precipitant solution containing DnifH Av1 was
much more than that in DnifE Av1 (Figs.2, 3). Perhaps it
seems to be led by the difference in the protein purity be-
tween the two proteins.
2.2 The selection of optimal composition in precipitant
solution
2.1.1 Hepes concentration Buffer is necessary for a
stability of pH value in protein solution. pH value is very
important for the electric charge on protein and the stabil-
ity of protein conformation, and the optimal pH value was
8.0-8.3 for crystallization for nitrogenase Av1, such as OP
Av1 and DnifZ Av1 (Mcpherson, 1983; Kim and Rees, 1992;
Huang et al., 2000). Since buffer, such as Hepes, is a kind of
salt affecting protein dissolution, it is necessary to select
its concentration. Like DnifE Av1, DnifH Av1 crystals grown
in the solution #4 for 28 d were the biggest in all of its
Acta Botanica Sinica 植物学报 Vol.46 No.1 200460
crystals. After only decrease of the Hepes concentration in
the solution #4 to that in the solution #3 (51 mmol/L), all of
its crystals formed was small. Unlike DnifH Av1, the crys-
tals of NifB- Av1 became to be larger after the decrease.
Table 2 also shows that the Hepes concentration had an
important effect on crystallization of DnifE Av1 and DnifH
Av1, although the optimal concentrations for the formation
of their bigger crystals were dependent on the change of
the method for the crystallization or other conditions.
2.1.2 PEG 8000 concentration As reported earlier
(Huang et al., 2000; 2001), the PEG concentration had an
important effect on the crystal number and crystal size of
both DnifH Av1 and DnifE Av1 (Tables 1, 3). From the re-
sults in Tables 1, 2, it is shown that the optimal PEG con-
centration for the crystallization of DnifH Av1 is about
5.50%. In comparison with DnifH Av1, the range of the
optimal PEG concentration for DnifE Av1 was somewhat
wider, 4.50%-5.50%.
Table 1 Differences in the crystallization among DnifH Av1, DnifE Av1 and NifB- Av1 by vapor diffusion in the hanging drop
method
Incubation Hepes conc.(1)
Solution(2)
Crystals
time (d) (mmol/L)
DnifH Av1(3) DnifE Av1(3) NifB-Av1(3)
Number Size(4) Number Size(4) Number Size(4)
9 #1 0 - 1 Bigger 0 -
50.84 #2 0 - 0 - 0 -
#3 0 - 2 Bigger 0 -
#4 0 - 3 Bigger 0 -
61.01 #5 0 - 1 Bigger 0 -
#6 0 - 0 - 0 -
71.18 #7 0 - 1 Small 0 -
81.35
#8 7 Small 11 Middle 3 Small
#9 0 - 0 - 0 -
#1 0 - 1 Big 0 -
50.84 #2 4 Middle 0 - 1 Small
#3 4 Small 2 Bigger 2 Big
#4 4 Bigger(5) 3 Big(6) 2 Small
28 61.01 #5 7 Middle 1 Big 1 Small
#6 0 - 0 - 0 -
71.18 #7 0 - 1 Small 0 -
81.35
#8 11 Middle(7) 6/5 Middle(8) 3 Small
#9 0 - 0 - 0 -
(1), Hepes concentration (pH 8.2-8.3) in the precipitant solutions containing glycerin was 11.57% (V/V); (2), in the precipitant solutions,
MgCl2 concentrations were 598.95 mmol/L in solution #1 to the solution #7, 275.99 mmol/L in solution #8 and 698.77 mmol/L in solution
#9; NaCl concentrations were 245.07 mmol/L in solutions #1, #2, #6, # 8 and #9 and 490.12 mmol/L in the others; PEG 8000 concentrations
(W/V) were 4.43% in solutions #1, #6, #8 and #9, 4.71% in solution #2, 5.00% in solutions #5 and #7, and 5.50% in solutions #3 and #4; (3),
the protein concentrations of DnifH Av1, DnifE Av1 and NifB- Av1 in 25 mmol/L Tris buffer (pH 7.4) containing 250 mmol/L NaCl were 8.
88 g/L, 11.48 g/L and 8.48 g/L, respectively; (4), the longest side of big, bigger, small and smaller crystal was ＞ 100 mm, ＜ 100 mm - ＞ 50
mm, ＜ 50 mm - ＞ 10 mm and ＜ 10 mm, respectively; (5), (6), (7) and (8), some crystals were observed after incubation for 28 d, shown in
Fig.2A-C and Fig.3A, B, respectively.
Table 2 Effect of Hepes and PEG 8000 concentrations on crystallization of DnifH Av1 and DnifE Av1 by liquid/liquid diffusion
method
Precipitant solution(1) Crystal(2)

PEG (%) Hepes (mmol/L)
DnifH Av1(3) DnifE Av1(3)
Number Size(4) Number Size(4)
50.69 6 Small 45 Small
5.00 61.01 >100 Small 2 Middle
71.18 1 Small 3/50 Middle/small
50.69 1/1 Middle/small 50 Small
5.50 61.01 7 Small 1/29 Middle/small
71.18 1/1 Middle/small 3/50 Middle/small
(1), the concentration of glycerin, NaCl and MgCl2 in the precipitant solution (pH 8.2-8.3) were 11.57% (V/V), 490.12 and 598.95
mmol/L, respectively; (2), the crystals were observed after incubation at 20 ℃ for 113 d; (3) and (4), the same as in Table 1.
BIAN Shao-Min et al.: Crystallization of MoFe Protein (DnifH Av1) from a nifH Deleted Strain of Azotobacter vinelandii 61
Table 3 Effects of PEG 8000 concentration on crystallization of DnifH Av1 and DnifE Av1 by liquid/liquid diffusion method
PEG Crystal(2)
(%)(1) DnifH Av1(3) DnifE Av1(3)
Number Size(4) Number Size(4)
3.79 50 Small 1/30 Middle/small
4.00 3 Small 3/3 Middle/small
4.21 2 Small 7/10 Middle/small
4.43 45 Small 6/20 Middle/small
4.71 30 Small 50 Small
(1), the concentration of glycerin, Hepes, NaCl and MgCl2 in the precipitant solution (pH 8.2-8.3) were 11.57% (V/V), 61.01, 245.06 and
598.95 mmol/L, respectively; (2), the same as in Table 2; (3) and (4), the same as in Table 1 .
Table 4 Effects of MgCl2 and NaCl concentrations on crystallization of DnifH Av1 and DnifE Av1 by liquid/liquid diffusion
method
Precipitant solution(1) Crystal(2)
NaCl MgCl2 DnifH Av1(3) DnifE Av1(3)
(mmol/L) (mmol/L) Number Size(4) Number Size(4)
275.99 50 Small 0 /
245.06
499.12 >100 Small 20 Small
598.95 45 Small 6/20 Middle/small
698.77 1/21 Middle/small 2 Middle
148.79 698.77 >100 Small 15 Small
(1), the concentrations of glycerin, Hepes, and PEG 8000 in the precipitant solution (pH 8.2-8.3) were 11.57% (V/V), 61.01 mmol/L and
4.43 % (W/V), respectively; (2)-(4), the same as in Table 2.
2.1.3 MgCl2 and NaCl concentration After the increase
of MgCl2 concentration in solution #8 to that in solution #9
in Table 1, crystals of either DnifH Av1 or DnifE
Av1disappeared. A few bigger crystals of the two proteins
appeared at the highest MgCl2 concentration (699 mmol/L)
in Table 4. But at the MgCl2 concentration, a lot of small-
sized crystals appeared when the NaCl concentration de-
creased from 245 mmol/L to 149 mmol/L (Table 4). This indi-
cates that the salts had also a significant effect on the crys-
tallization of DnifH Av1 and DnifE Av1.
3 Discussion
Because the DnifH Av1 crystals picked from the precipi-
tant solution were not washed before its dissolution with
the buffer containing 0.3 mmol/L NaCl, it is possible for the
crystalline DnifH Av1 to be contaminated by the precipi-
tant solution, leading to slightly change its behavior on
SDS-PAGE gel. But the bands on the gel were similar to
those of OP Av1 (Fig.1). And in comparison with the DnifH
Av1 before crystallization, the crystalline DnifH Av1 was
much purer. Since the protein crystallization is one of meth-
ods for protein purification, the phenomenon could happen.
Hence, this indicates that the crystal could be one of DnifH
Av1.
In the precipitant solution suited for DnifH Av1 and
DnifE Av1 to be crystallized, it is possible that the contami-
nation protein in DnifH Av1 was not crystallized and
dissolved, leading to a formation of a lot of protein pellet.
Since the contamination protein in DnifH Av1 solution was
much more than those in DnifE Av1 solution, more pellet
could appear in the precipitant solution containing DnifH
Av1 solution (Figs.2, 3). However, DnifH Av1 was similar in
the time for crystal formation to DnifB Av1 with a better
purity. This indicates that a main factor affecting time of
crystal formation seems to be protein kind, rather than pro-
tein purity; and protein purity could significantly be re-
lated to the amount of protein pellet.
Under some conditions, the time for formation of DnifH
Av1 was similar to that of NifB- Av1, but longer than that of
DnifE Av1. And under some conditions, the optimal con-
centration of a chemical mentioned above for crystalliza-
tion of DnifH Av1 was similar to those of DnifE Av1, but
different from those of NifB- Av1. Therefore, it is suggested
that the feature of DnifH Av1 could be different from that of
either DnifE Av1 or NifB- Av1.
References:
Allen R M, Chatterjee R, Ludden P W, Shah V K. 1995. Incorpo-
ration of iron and sulfur from NifB cofactor into the iron-
molybdenum cofactor of nitrogenase. J Biol Chem, 270:
26890-26896.
Burgess B K, Lowe D J. 1996. Mechanism of molybdenum
nitrogenase. Chem Rev, 96:2983-3011.
Gavini N, Ma L, Watt G, Burgess B K. 1994. Purification and
Acta Botanica Sinica 植物学报 Vol.46 No.1 200462
characterization of a FeMo cofactor-deficient MoFe protein.
Biochemistry, 33:11842-11849.
Huang J-F , Wang Y-P, Dong Z-G, Huang X-M, Wang D-Y, Lü Y-
B , Wang Z-P. 2000. Studies on crystalline growth of MoFe
protein from a nifZ deleted strain of Azotobacter vinelandii.
Acta Bot Sin , 42:383-387.
Huang J-F , Dong Z-G , Wang D-Y, Lü Y-B, Zhang H-F, Wang Y-
P , Han Y , Dai X-H. 2001. Growth of the crystals of nitroge-
nase MnFe protein. Acta Bot Sin , 43: 375-379.
Kim J, Rees D C. 1992. Structure models for the metal centers in
the nitrogenase molybdenum-iron protein. Science, 257:1677-
1682.
Lü Y-B, Zhao Y, Zhao J-F, Dai X H, Cang H-X, Wang Y-P, Huang
J-F. 2003. Growth of large single crystals of nitrogenase CrFe
protein and MnFe protein. Acta Bot Sin, 45:289-294.
Mcpherson A J. 1983. Group of Crystalline Growth, Institute of
Biophysics, The Chinese Academy of Sciences trans. The
growth and preliminary investigation of protein nucleic acid
crystals for X-ray diffraction analysis. Glick D. Methods of
Biochemical Analysis. Vol.23. Beijing: Chinese Science and
Technology Press. 249-345. (in Chinese)
Rangaraj P, Ryle M J, Lanzilotta W N, Ludden P W, Shah V K.
1999. In vivo biosynthesis of iron-molybdenum cofactor and
maturation of the nif-encoded apodinitrogenase. J Biol Chem,
274:19778-19784.
Ribbe M W, Hu Y, Guo M, Schmid B, Burgess B. 2002. The
FeMoco-deficient MoFe protein produced by a nifH deletion
strain of Azotobacter vinelandii shows unusual P-cluster
Features. J Biol Chem, 277:23469-23476.
Brigle K E, Weiss M C, Newton W E, Dean D R. 1987. Products
of the iron-molybdenum cofactor-specific biosynthetic genes,
nifE and nifN, are structurally homologous to the products of
nitrogenase molybdenum-iron protein genes, nifD and nifK. J
Bacteriol, 169:1547-1553.
Schmid B, Ribbe M W, Einsle O, Yoshida M, Thomas L M, Dean
D R, Rees D C, Burgess B K. 2002. Structure of a cofactor-
deficient nitrogenase MoFe protein. Science, 296:352-356.
Tal S, Chun T W, Gavini N, Burgess B. 1991. The DnifB (or
DnifE) FeMoco-deficient MoFe protein is different from the
DnifH protein. J Biol Chem, 266:10654-10657.
Zhao J-F , Zhao Y, Wang Z-P,Lü Y-B, Qian Z-X, Huang J-F.
2003a. Purification and activation in vitro of MoFe Protein
from nifE deleted mutant strain of Azotobacter vinelandii. Acta
Bot Sin,45:815-819.
Zhao Y, Zhao J-F , LÜ Y-B, Wang Z-P,Wang Y-P, Huang J-F.
2003b. Crystallization of nitrogenase MoFe protein (DnifE
Av1) from nifE deleted mutant strain of Azotobacter vinelandii.
Acta Bot Sin , 45:427-431.
(Managing editor: HE Ping)