Substitution of histidine for arginine-101 of dinitrogenase reductase disrupts electron transfer to dinitrogenase

Lowery, Robert G.; ␣Chang, Christina L.; Davis, Lawrence C.; McKenna, M. C.; Stephens, Philip J.; Ludden, Paul W.

doi:10.1021/bi00429a038

Cited by 55 publications

(30 citation statements)

References 35 publications

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“…These might include an initial docking complex, which further aligns to create a state competent for MgATP hydrolysis, which would be followed by a state competent for electron transfer. Similar models have been proposed for FeP and MoFeP interactions (Lowery et al, 1989;Howard, 1993).…”

Section: Discussionmentioning

confidence: 67%

“…Earlier work with heterologous FeP-MoFeP complexes showed that such complexes would hydrolyze MgATP, but would not transfer electrons (Smith et al, 1976;Emerich & Burris, 1978). Likewise, changing Arg 100 of the FeP was found to uncouple MgATP hydrolysis from intercomponent electron transfer (Lowery et al, 1989;Wolle et al, 1992b). The data presented in this work show that Arg 140 and Lys 143 of the FeP and their interaction with the MoFeP are critical for coupling MgATP hydrolysis to electron transfer.…”

Section: Discussionmentioning

confidence: 99%

“…An analysis of the FeP crystal structure (Georgiadis et al, 1992) reveals at least 3 stretches of amino acids that might be involved (Chang et al, 1988;Lowery et al, 1989;Willing & Howard, 1990;Wolle et al, 1992b). In addition, Arg 100 is the site of ADP-ribosylation, a posttranslation modification mechanism used to regulate nitrogenase activity (Ludden & Roberts, 1989).…”

Section: Site-directed Mutagenesis Of the A Vinelandii Nitrogenase Fepmentioning

confidence: 99%

“…Models for how these 2 proteins might dock for electron transfer have been suggested from analysis of the individual structures and from the position of the respective metal centers (Kim et al, 1992;Howard, 1993;Rees et al, 1993;Kim & Rees, 1994). In addition, chemical crosslinking studies (Willing et al, 1989;Willing & Howard, 1990) and site-directed mutagenesis studies of amino acids on both the FeP and the MoFeP have implicated Arg 100 (Chang et al, 1988;Lowery et al, 1989;Wolle et al, 199213) and Glu 112 (Willing & Howard, 1990) of the FeP and MoFeP a-subunit Asp 161 (Kim et al, 1992) and &subunit Phe 125 (Fisher et al, 1992) in docking. In the present work, data are presented revealing that the 2 positively charged amino acids, Arg 140 and Lys 143, of A .…”

mentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Site-directed Mutagenesis Of the A Vinelandii Nitrogenase Fepmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…The most striking example of this phenomenon is illustrated by the observation that the ␣-Ser 69 substitution within the MoFe protein effectively suppresses the acetylenesensitive phenotype of a mutant strain that has leucine substituted for the Fe protein Arg 100 residue. The Fe protein Arg 100 residue is located at the component protein-docking interface (34,35), and substitutions at this position have been shown to affect intercomponent electron transfer (20,36). The general ability of the ␣-Ser 69 substitution to suppress the acetylenesensitive phenotype is best explained by a structural barrier that prevents acetylene from effectively binding to the active site of the altered protein.…”

Section: Nature Of the Acetylene Resistance Phenotype Elicited By Thementioning

confidence: 99%

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Christiansen¹,

Cash²,

Seefeldt³

et al. 2000

Journal of Biological Chemistry

111

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A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene. Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the ␣-subunit Gly 69 residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the ␣-Ser 69 substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the ␣-subunit Val 70 residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity.

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Nitrogen Fixation

Newton¹

2000

Kirk-Othmer Encyclopedia of Chemical Technology

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Nitrogen fixation converts inert atmospheric molecular nitrogen gas into reduced forms whereby the nitrogen may be used by all life forms for protein and nucleic acid production. The biological process occurs only in microorganisms and is the principal contributor of fixed nitrogen to the biosphere. Fixed nitrogen is also derived from nonbiological processes, such as fires, volcanoes, and lightning, and from industrial fixation. The Haber‐Bosch ammonia process is highly energy‐intensive but is the most economical industrial process available. Agriculturally, the most important source of biologically fixed nitrogen is the symbiotic system involving leguminous plants which harbor rhizobia bacteria in nodules on their roots. Smaller contributions are made by other, less formal systems and by free‐living microbes. All of the biological systems use a similar metalloenzyme complex, called nitrogenase. Nitrogen fixation research is discussed from a genetic standpoint, as well as in terms of model chemistry and economic advantages of the biological system. The chemical approaches aimed at duplicating the biological process are presented.

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Substitution of histidine for arginine-101 of dinitrogenase reductase disrupts electron transfer to dinitrogenase

Cited by 55 publications

References 35 publications

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

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

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Nitrogen Fixation

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