On the structure and function of nitrogenase from Clostridium pasteurianum W5

Zumft, Walter G.; Cretney, Walter C.; Huang, Tao; Mortenson, Leonard E.; Palmer, Graham

doi:10.1016/0006-291x(72)90887-x

Cited by 53 publications

(37 citation statements)

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“…In the present work we describe the current fractionation of molybdoferredoxin and report on the nature and physicochemical properties of the inactive species with resonance a t g = 1.94. The consequence of our findings will influence previously reported physicochemical data of molydoferredoxin ; preliminary reports have appeared [8,11].…”

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confidence: 55%

Evidence for a Catalytic‐Centre Heterogeneity of Molybdoferredoxin from Clostridium pasteurianum

Zumft

Mortenson

1973

European Journal of Biochemistry

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Molybdoferredoxin, the high-molecular-weight component of clostridial nitrogenase has been separated into two components by gradient chromatography on DEAE-cellulose. One component when combined with azoferredoxin, the low-molecular-weight component of nitrogenase, does not reduce acetylene, evolve hydrogen from dithionite or hydrolyse ATP ; the other does. Compared with the active component, the inactive species lacks molybdenum and approximately two thirds of the iron. Both species can be identified by their electron paramagnetic resonance spectra. Active molybdoferredoxin has resonances at g = 4.27, 3.78 and 2.01, whereas the inactive species has signals a t g = 2.05 and 1.92. Apart from the difference in their catalytic centre, the protein moiety of both species seems to be identical as based on subunit composition, sedimentation and electrophoretic behaviour, amino acid composition and immunological reaction with a molybdoferredoxin antiserum. The method currently employed for purification of nitrogenase proteins and the fractionation of molybdoferredoxin is described. The consequence of this finding influences previously reported physico-chemical data of molybdoferredoxin.Molybdoferredoxin (molybdenum-iron protein), the high-molecular-weight component of nitrogenase, has been purified from a variety of organisms and its physico-chemical properties have been reported [ 1-51. There exists, however, considerable disagreement on the protein characteristics, not only from different organisms, but also in and between laboratories using the same starting material [I, 2 , 5 , 6 ] .This led us to question the homogeneity of various molybdoferredoxin preparations rather than attribute the differences to biological variation, although this latter possibility can not be disregarded completely. We will present evidence in this paper that molybdoferredoxin from Clostridium pasteurianum consists of at least two components. One is catalytically active when reconstituted with azoferredoxin (iron protein), the low-molecular-weight component of nitrogenase. 22-24 atoms of iron and acid-labile sulfides per molecular weight of 220000 [5]. The protein moiety of the other species seems to be identical with native molybdoferredoxin ; however, the molecule is devoid of molybdenum and has a low iron content. No catalytic activity could be demonstrated upon reconstitution with azoferredoxin.Electron paramagnetic resonance (EPR) spectroscopy has become a useful tool in the investigation of nitrogenase [2-4,7--111. To date, electron paramagnetic resonance spectroscopy is the only way to identify the presence of the inactive molybdoferredoxin species. It is also from EPR data t h a t we conclude the occurrence of this second species in moly bdoferredoxin preparation of other provenience [2-41. While this work was in preparation, Davis et al. [7] found that the previously reported resonance at g = 1.94 of the molybdenum-iron protein of Azotobacter vinelandii is not present in highly purified samples and suggested that it might be a c...

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confidence: 55%

Evidence for a Catalytic‐Centre Heterogeneity of Molybdoferredoxin from Clostridium pasteurianum

Zumft

Mortenson

1973

European Journal of Biochemistry

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“…This state would be analogous to the heterologous Clostridium pasteurianurn FeP-A. vinelandii MoFeP complex that catalyzes MgATP hydrolysis, but not electron transfer (Smith et al, 1976;Emerich & Burris, 1978 (Orme-Johnson et al, 1972;Zumft et al, 1972), a change in the midpoint potential of the cluster by -150 mV (Watt et al, 1986), changes in the CD spectrum (Stephens et al, 1979), shifts in the NMR resonances of protons magnetically coupled to the paramagnetic [4Fe-4S] cluster (Meyer et al, 1988), changes in scattering of X-rays (Chen et al, 1994), and in Fez+ chelation (Walker & Mortenson, 1974). This conformation of the FeP would be required for the formation of the second, MgATP hydrolytic state, with the MoFeP.…”

Section: Discussionmentioning

confidence: 99%

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Seefeldt

1994

Protein Science

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Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum-iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site-directed mutagenesis. The altered FePs were expressed in A . vinelandii in place of the wild-type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild-type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments. In addition, it was found that changing Arg 140 and Lys 143 to Gln resulted in a significant uncoupling of MgATP hydrolysis from intercomponent electron transfer rates between FeP and MoFeP. The wild-type complex was found to hydrolyze 2.5 MgATP per electron transferred, whereas the R140Q and K143Q proteins, when coupled to wild-type MoFeP, required 6 and 31 MgATP for each electron transferred, respectively. It is concluded that both Arg 140 and Lys 143 of the A . vinelandii nitrogenase FeP are important in the docking interaction with the MoFeP and that these residues probably function in aligning the FeP with the MoFeP for intercomponent electron transfer. A model is discussed in which these positively charged amino acids of the FeP might interact with negatively charged amino acids (Asp and Glu) on the MoFeP near its 8Fe cluster.Keywords: electron transfer; iron protein; molybdenum-iron protein; MgATP hydrolysis; nitrogenase; protein docking; site-directed mutagenesis Biological dinitrogen reduction is catalyzed by nitrogenase, a complex metalloenzyme consisting of the 2 separable component proteins, the molybdenum-iron protein and the iron protein (current reviews: Burris, 1991;Smith & Eady, 1992;Dean et al., Reprint requests to: Lance C . Seefeldt, Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300; e-mail: seefeldt@cc.usu.edu.Abbreviations: FeP, nitrogenase iron protein; MoFeP, nitrogenase molybdenum-iron protein; FeP-MoFeP, iron protein-molybdenumiron protein comple...

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“…Whether activation of inactive nitrogenase proteins is continually taking place in fully derepressed populations remains to be tested critically. During purification of nitrogenase from Clostridium pasteurianum an inactive derivative of the iron-molybdenum protein which lacks molybdenum and much of its normal complement of iron has been separated (Zumft, Cretney, Huang, Mortenson & Palmer, 1972). If such a species occurs in vivo, reactivation in the absence of further protein synthesis may be possible.…”

Section: Discussionmentioning

confidence: 99%

Control of Nitrogenase Synthesis in Klebsiella pneumoniae

Tubb

Postgate

1973

Journal of General Microbiology

103

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SUMMARYSulphate-limited continuous cultures of Klebsiella pneumoniae showed a proportional repression of nitrogenase activity with increasing concentrations of ammonium ion in the influent medium. A fully repressed population had a 50 % greater bacterial density than a fully derepressed one. On derepression, synthesis of nitrogenase lagged for 90 min after exhaustion of NH,+ from the medium but was complete within one doubling time. Casamino acids did not decrease the prefixation lag obtained under sulphate-limited conditions in the chemostat. Longer lags were obtained when NH,+ was exhausted under N-limited conditions. Such lags were decreased by Casamino acids, yeast extract, or L-aspartate. Aspartate-N completely repressed nitrogenase under sulphate-or carbon-limited conditions but not under nitrogen limitation. In the initial stages of repression of nitrogenase synthesis by NH,+, apparent production of active enzyme continued for a short time in the absence of protein synthesis de novo; nitrogenase was then diluted out as the organisms multiplied. Repression by NH,+ was at the level of mRNA transcription according to studies with rifampicin and chloramphenicol. ' Coding capacity' for nitrogenase synthesis declined with a half-life of about 4.5 min following inhibition of RNA synthesis with rifampicin. NH,+ did not influence the decay rate but stimulated translation of such nitrogenase-specifying mRNA as had been initiated.

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On the structure and function of nitrogenase from Clostridium pasteurianum W5

Cited by 53 publications

References 9 publications

Evidence for a Catalytic‐Centre Heterogeneity of Molybdoferredoxin from Clostridium pasteurianum

Evidence for a Catalytic‐Centre Heterogeneity of Molybdoferredoxin from Clostridium pasteurianum

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Control of Nitrogenase Synthesis in Klebsiella pneumoniae

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