Catalytic and Biophysical Properties of a Nitrogenase Apo-MoFe Protein Produced by a nifB-Deletion Mutant of Azotobacter vinelandii

Christiansen, J. A.; Goodwin, Paul J.; Lanzilotta, William N.; Seefeldt, Lance C.; Dean, Dennis R.

doi:10.1021/bi981165b

Cited by 196 publications

(304 citation statements)

References 62 publications

Supporting

Mentioning

282

Contrasting

Unclassified

Order By: Relevance

“…15 N-labeled hydrazine was obtained from Cambridge Isotopes (Andover, MA). Azotobacter vinelandii strains DJ995 (wild-type MoFe protein), DJ1310 (α-70 Ala MoFe protein), DJ997 (α-195 Gln MoFe protein), and DJ1316 (α-195 Gln / α-70 Ala MoFe protein) were constructed and nitrogenase proteins were expressed and purified as described previously (31). All proteins used were greater than 95% pure as judged by SDS-PAGE analysis using Coomassie blue staining.…”

Section: Materials and Proteinsmentioning

confidence: 99%

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

et al. 2007

Self Cite

View full text Add to dashboard Cite

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N 2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H 2 N-NH 2 ). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH 3 . Diazene reduction, like N 2 reduction, is inhibited by H 2 . This contrasts with the lack of H 2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N 2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with α-195 His substituted by Gln and α-70 Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3/2 resting state FeMo-cofactor to a novel S = 1/2 state with g 1 = 2.09, g 2 = 2.01, and g 3 ~ 1.98. 15 N-and 1 H-ENDOR establish that this state consists of a diazene-derived [-NH x ] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NH x ] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N 2 -reduction pathway, and that the diazene-and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N 2 by nitrogenase. The implications of these findings for the mechanism of N 2 reduction by nitrogenase are discussed. KeywordsFeMo-cofactor; EPR; ENDOR; Active Site; Substrate Nitrogenase is the enzyme responsible for catalyzing biological reduction of N 2 to two NH 3 , an essential reaction in the global biogeochemical nitrogen cycle (1-3). The minimum stoichiometry for the nitrogenase catalyzed reduction of N 2 involves delivery of 8e − and 8H + (eqn 1). † This work was supported by grants from the National Institutes of Health (R01-GM59087 to LCS and DRD; HL13531 to BMH), the National Science Foundation (MCB-0316038 to BMH) and the United States Department of Agriculture Postdoctoral Fellowship program (2004-35318-14905 to BMB).*Address correspondence to these authors: LCS, phone (435) 797-3964, fax (435) email seefeldt@cc.usu.edu; DRD, phone (540) 231-5895, fax (540) email deandr@vt.edu; BMH, phone (847) (Figure 1).Relatively little is known at a molecular level about the nitrogenase N 2 -reduction mechanism beyond the fact that N 2 binds to and is reduced at one or more of the metal atoms of FeMocofactor ( Figure 1) Both the Chatt and Schrock cycles belong to one fundamental class of potential nitrogenase mechanismsin which the first three 'H-atoms' (e − /H + ) are sequentially added to a single N atom, in those instances the distal N of an end-on bound N 2 , followed by cleava...

show abstract

Section: Materials and Proteinsmentioning

confidence: 99%

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

et al. 2007

Self Cite

View full text Add to dashboard Cite

show abstract

“…Wild-type MoFe protein was isolated from A. vinelandii strain DJ995 [23], α-70 Ala MoFe protein from DJ1310 [19], α-70 Gly MoFe protein from DJ1313, α-191 Ala MoFe protein from DJ1242, and α-70 Ala /α-191 Ala MoFe protein from DJ1495. All MoFe proteins in this study contained a poly-histidine insertion near the carboxyl terminus of each α-subunit.…”

Section: Methodsmentioning

confidence: 99%

“…A. vinelandii strains DJ1242, DJ1313, and DJ1495 were constructed for this study, and a detailed description of strain construction follows. DJ1313 was constructed by transforming DJ995 [23] with pDB1133 (SstI 'nifH-nifD'fragment containing GGG as codon 70 of nifD). The transformants were selected by loss of the ability to grow under nitrogen fixing conditions.…”

Section: Methodsmentioning

confidence: 99%

“…This strain was selected by a slower growth rate under nitrogen fixing conditions. For protein purification, cells were grown at 30°C in 120 L culture as described previously [23]. Cell-free extracts were prepared by an osmotic shock method and the MoFe protein was purified by an immobilized metal-affinity chelation chromatography (IMAC) protocol [23].…”

Section: Methodsmentioning

confidence: 99%

“…For protein purification, cells were grown at 30°C in 120 L culture as described previously [23]. Cell-free extracts were prepared by an osmotic shock method and the MoFe protein was purified by an immobilized metal-affinity chelation chromatography (IMAC) protocol [23]. The Fe protein used in all experiments was purified from wild-type cells and did not contain a histidine tag.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Alkyne substrate interaction within the nitrogenase MoFe protein

Santos

Mayer

Barney

et al. 2007

Journal of Inorganic Biochemistry

Self Cite

View full text Add to dashboard Cite

Nitrogenase catalyzes the biological reduction of N 2 to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC≡CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein α-70 Val residue, whose side-chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of α-70 Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC≡C-CH 3 ), whereas substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the α-70 Val residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC≡C-CH 2 -CH 3 ). This protein shows no detectable reduction of the internal alkyne 2-butyne (H 3 C-C≡C-CH 3 ). In contrast, substitution of the nearby α-191 Gln residue by alanine, in combination with the α-70 Ala substitution, does result in significant reduction 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves sideon binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein.

show abstract

Nitrogenase

Schmid

Chiu

Ramakrishnan

et al. 2004

Handbook of Metalloproteins

View full text Add to dashboard Cite

Nitrogenase catalyzes the enzymatic reduction of atmospheric dinitrogen to ammonia during the process of biological nitrogen fixation. Nitrogenase consists of two component metalloproteins, the iron (Fe‐) protein and the molybdenum‐iron (MoFe‐) protein, that together mediate the ATP‐hydrolysis–dependent reduction of substrates to products. Crystallographic studies have established the structures of the component proteins and the associated complex metallocenters of nitrogenase, including the iron‐molybdenum cofactor that provides the active site for substrate reduction and the P‐cluster that participates in electron transfer between the Fe‐protein and MoFe‐protein. Striking parallels are evident in the interaction of the nucleotides with the Fe‐protein and with a broad class of nucleotide‐binding proteins involved in signal and energy transduction processes. Together with kinetic, spectroscopic, and synthetic model compound studies, these structures provide a framework for addressing the mechanism of substrate reduction by nitrogenase.

show abstract

Catalytic and Biophysical Properties of a Nitrogenase Apo-MoFe Protein Produced by a nifB-Deletion Mutant of Azotobacter vinelandii

Cited by 196 publications

References 62 publications

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase

Contact Info

Product

Resources

About

Catalytic and Biophysical Properties of a Nitrogenase Apo-MoFe Protein Produced by a nifB-Deletion Mutant of Azotobacter vinelandii

Cited by 196 publications

References 62 publications

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N2 Reduction

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N2 Reduction

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase

Contact Info

Product

Resources

About

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction