Purification of the nitrogenase proteins from Clostridium pasteurianum

Tso, Man-Yin W.; Ljones, Torbjørn; Burris, R. H.

doi:10.1016/0005-2728(72)90193-4

Cited by 59 publications

(29 citation statements)

References 7 publications

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“…An examination of the protein sequencing data showed that the purified Fe protein could not contain more than trace amounts of proteins from the nifH-like genes (Wang et al, 1988). However, this conclusion does not rule out the possibility that other NifH-related proteins are present in the cell but are separated from the nifH1-encoded (Tso et al, 1972;Zumft & Mortenson, 1973).…”

Section: Discussionmentioning

confidence: 95%

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Kasap

Chen

2005

Microbiology

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Previous studies identified five nifH-like genes (nifH2 through nifH6) in Clostridium pasteurianum (strain W5), where the nifH1 gene encodes the nitrogenase iron protein. Transcripts of these nifH genes, with the exception of nifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by the nifH-like genes were not reported. The nifH2 and nifH6 genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region of nifH2 and nifH6 was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of the nif genes of single-colony isolates of C. pasteurianum suggest that the nifH6 designation should be eliminated. Here the size of mRNA from nifH2 and the detection of the NifH2 polypeptide in nitrogen-fixing cells of C. pasteurianum are reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that the nifH1 and nifH2 mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH of Azotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixing C. pasteurianum cells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixing C. pasteurianum cells.

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Section: Discussionmentioning

confidence: 95%

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Kasap

Chen

2005

Microbiology

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“…Clostridium pasteurianum strain W5 was a gift from Professor J. S. Chen (Virginia Polytechnic Institute and State University) and was grown as described (18). Nitrogenase Fe and MoFe proteins were purified from each bacterial source as described (19) to apparent homogeneity as judged by migration on SDS-polyacrylamide gel with Coomassie Blue staining (20).…”

Section: Methodsmentioning

confidence: 99%

Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

Ryle¹,

Seefeldt

2000

Journal of Biological Chemistry

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The hydrolysis of ATP to ADP and P i is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron ( The hydrolysis of ATP to ADP and P i has long been known to be an integral part of all nitrogenase substrate reduction reactions (1-4). The stoichiometry for coupling ATP hydrolysis to substrate reduction varies depending on reactions conditions, with a minimum of 2 ATP molecules hydrolyzed for each electron transferred to substrate (5, 6). Thus, the reduction of N 2 to 2NH 3 by nitrogenase requires the hydrolysis of at least 16 ATP molecules (Equation 1). There have been isolated examinations of the interactions of nucleotides other than adenine nucleotides with nitrogenase. To summarize, no nucleotide other than ATP has been shown to support substrate reduction, although there is a recent report that protons can be reduced at extremely low rates without ATP within an Fe protein-MoFe protein complex created with AlF 4 Ϫ ⅐ADP (10). The hydrolysis of ATP to ADP and P i is the predominant reaction observed. Apparently, the MoFe protein alone or the Fe protein-MoFe protein complex can catalyze the further hydrolysis of ADP at extremely low rates (11,12), although these reactions do not appear to be part of the substrate reduction mechanism. A divalent metal-bound form of ATP is required for hydrolysis, with MgATP showing the highest rates of substrate reduction (13). There is, however, evidence suggesting that nucleotides other than adenosine di-or triphosphates interact with nitrogenase. The Fe protein is known to bind 2 molecules of either MgATP or MgADP, with each resulting in distinct protein conformational changes (1). These protein conformational changes have been detected by a variety of methods. For example, the line shape of the EPR signal of the S ϭ 1/2 state of the reduced 1ϩ cluster of the Fe protein changes upon MgATP or MgADP binding (14). MgGTP was observed to induce small changes in the EPR line shape of the Fe protein, suggesting that MgGTP binds to the Fe protein and induces at least partial protein conformational changes (14). Another technique that reports protein conformational changes in the Fe protein is the sensitivity of the [4Fe-4S] cluster to chelation by iron-specific chelators like bathophenanthroline or ␣,␣Ј-dipyridyl. In the absence of nucleotides, the [4Fe-4S] cluster of the Fe protein is resistant to chelation by chelators (15, 16). Upon binding MgATP, however, a timedependent release of all of the iron atoms from the cluster to

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“…Nitrogenase is a two-component metalloenzyme complex that catalyzes the ATP-dependent reduction of dinitrogen to ammonia (nitrogen fixation) (2,11,21,52). The fixation of atmospheric nitrogen is an essential process for the survival of life on earth and a critical part of the global nitrogen cycle (14,22,39).…”

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confidence: 99%