Catalytic and biophysical properties of a nitrogenase apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii

被引:163
作者
Christiansen, J
Goodwin, PJ
Lanzilotta, WN
Seefeldt, LC [1 ]
Dean, DR
机构
[1] Utah State Univ, Dept Chem & Biochem, Logan, UT 84322 USA
[2] Virginia Polytech Inst & State Univ, Dept Biochem, Fralin Biotechnol Ctr, Blacksburg, VA 24061 USA
关键词
D O I
10.1021/bi981165b
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A Zn-immobilized metal-affinity chromatography technique was used to purify a poly-histidine-tagged, FeMo-cofactorless MoFe protein (apo-MoFe protein) from a nifB-deletion mutant of Azotobacter vinelandii. Apo-MoFe protein prepared in this way was obtained in sufficient concentrations for detailed catalytic, kinetic, and spectroscopic analyses. Metal analysis and electron paramagnetic resonance spectroscopy (EPR) were used to show that the apo-MoFe protein does not contain FeMo-cofactor. The EPR of the as-isolated apo-MoFe protein is featureless except for a minor S = 1/2 signal probably arising from the presence of either a damaged P cluster or a P cluster precursor. The apo-MoFe protein has an alpha(2)beta(2) subunit composition and can be activated to 80% of the theoretical MoFe protein value by the addition of isolated FeMo-cofactor. Oxidation of the as-isolated apo-MoFe protein by indigodisulfonate was used to elicit the parallel mode EPR signal indicative of the two-electron oxidized form of the P cluster (P2+). The midpoint potential of the p(N)/P2+ redox couple for the apo-MoFe protein was shown to be shifted by -63 mV when compared to the same redox couple for the intact MoFe protein. Although the apo-MoFe protein is not able to catalyze the reduction of substrates under turnover conditions, it does support the hydrolysis of MgATP at 60% of the rate supported by the MoFe protein when incubated in the presence of Fe protein. The ability of the apo-MoFe protein to specifically interact with the Fe protein was also shown by stopped-flow techniques and by formation of an apo-MoFe protein-Fe protein complex. Finally, the two-electron oxidized form of the apo-MoFe protein could be reduced to the one-electron oxidized form (P1+) in a reaction that required Fe protein and MgATP. These results are interpreted to indicate that the apo-MoFe protein produced in a nifB-deficient genetic backround contains intact P clusters and P cluster polypeptide environments. Small changes in the electronic properties of P clusters contained within the apo-MoFe protein are most Likely caused by slight perturbations in their polypeptide environments.
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页码:12611 / 12623
页数:13
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共 70 条
[1]   Isolation of two forms of the nitrogenase VFe protein from Azotobacter vinelandii [J].
Blanchard, CZ ;
Hales, BJ .
BIOCHEMISTRY, 1996, 35 (02) :472-478
[2]   COMPLETE NUCLEOTIDE-SEQUENCE OF THE AZOTOBACTER-VINELANDII NITROGENASE STRUCTURAL GENE-CLUSTER [J].
BRIGLE, KE ;
NEWTON, WE ;
DEAN, DR .
GENE, 1985, 37 (1-3) :37-44
[3]   LARGE-SCALE PURIFICATION OF HIGH-ACTIVITY AZOTOBACTER-VINELANDII NITROGENASE [J].
BURGESS, BK ;
JACOBS, DB ;
STIEFEL, EI .
BIOCHIMICA ET BIOPHYSICA ACTA, 1980, 614 (01) :196-209
[4]   THE IRON MOLYBDENUM COFACTOR OF NITROGENASE [J].
BURGESS, BK .
CHEMICAL REVIEWS, 1990, 90 (08) :1377-1406
[5]  
BURGESS BK, 1996, CHEM REV, P96
[6]  
CHROMY V, 1974, CLIN CHEM, V20, P1362
[7]   NITROGENASE METALLOCLUSTERS - STRUCTURES, ORGANIZATION, AND SYNTHESIS [J].
DEAN, DR ;
BOLIN, JT ;
ZHENG, LM .
JOURNAL OF BACTERIOLOGY, 1993, 175 (21) :6737-6744
[8]  
DEITS TL, 1990, J BIOL CHEM, V265, P3859
[9]  
Dutton P L, 1978, Methods Enzymol, V54, P411
[10]   Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase [J].
Duyvis, MG ;
Wassink, H ;
Haaker, H .
FEBS LETTERS, 1996, 380 (03) :233-236