<i>Azotobacter vinelandii</i> Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on the Catalyzed Reduction of Acetylene and Ethylene

Fisher, Karl; Dilworth, M. J.; Kim, Chul‐Hwan; Newton, William E.

doi:10.1021/bi992092e

Cited by 55 publications

(106 citation statements)

References 46 publications

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“…7. We have studied the effects of substitutions at these two positions on nitrogenase function in some detail previously [14,26,27,[32][33][34]43,44]. This information led us to chose the aH195Q, aH195N and aQ191K MoFe proteins as suitable vehicles to assist in determining the potential function(s) of the three-electron-reduced FeMo-cofactor-based species responsible for the new S = 3/2 signal that we call signal 1b.…”

Section: Discussionmentioning

confidence: 99%

“…The growth of wild-type (a-191 Gln /a-195 His ), DJ255 (a-191 Lys /a-195 His ), DJ178 (a-191 Gln /a-195 Asn ), and DJ540 (a-191 Gln /a-195 Gln ) strains of A. vinelandii, nitrogenase derepression, cell-extract preparation, purification of the nitrogenase MoFe protein component, and exchange into 25 mM HEPES (pH 7.4) were performed as previously described [32][33][34]. The resulting MoFe protein specific activities were 2600 (wild-type), 1500 (aQ191K), 1500 (aH195N), and 2700 (aH195Q) nmol H 2 (min mg MoFe protein) À1 , under 101 kPa Ar in the presence of a 20-fold molar excess of wild-type Fe protein with an ATP/2e À value of 5.1 ± 0.5.…”

Section: Cell Growth and Protein Purificationmentioning

confidence: 99%

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Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Fisher

Lowe

Tavares

et al. 2007

Journal of Inorganic Biochemistry

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Section: Discussionmentioning

confidence: 99%

Section: Cell Growth and Protein Purificationmentioning

confidence: 99%

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Fisher

Lowe

Tavares

et al. 2007

Journal of Inorganic Biochemistry

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“…These are of particular interest as they bind, but do not reduce N 2 well or at all respectively. and it has been suggested that the α-H195 residue plays a role in channelling protons to the FeMo-cofactor active site [15] .…”

Section: Introductionmentioning

confidence: 99%

Photolysis of Hi‐CO Nitrogenase – Observation of a Plethora of Distinct CO Species Using Infrared Spectroscopy

Yan¹,

Dapper

George

et al. 2011

Eur J Inorg Chem

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Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii nitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm−1 and 1679 cm−1 together with positive bands at 1711 cm−1, 2135 and 2123 cm−1. The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm−1 is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species ‘Hi-1’ and ‘Lo-1’ respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol−1. Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative ‘Hi-2’ bands at 1936 and 1858 cm−1 with a positive ‘Lo-2’ band at 1780 cm−1, while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative ‘Hi-3’ bands at 1938 and 1911 cm−1 and a positive feature ‘Lo-3’ band at 1921 cm−1. These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo-cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.

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“…Earlier work on wild-type nitrogenase revealed that proton addition to acetylene during its reduction to ethylene occurs with stereospecificity [27][28][29]. The product of acetylene (HC≡CH) reduction in the presence of D 2 O is 96 % cis-ethylene (HDC=CDH), with only 4 % of the trans-isomer [30]. This stereospecificity would be consistent with side-on binding of acetylene to one or more Fe atoms of FeMo-cofactor, with both protons (or deuterons) added to one side of the bound acetylene.…”

Section: Discussionmentioning

confidence: 99%

“…Ed Stiefel suggested a concerted proton and electron addition mechanism to explain this stereospecificity of proton addition to acetylene [31]. Interestingly, it was later found that lowering the electron flux through nitrogenase or substituting certain amino acids around FeMo-cofactor, can relax this stereospecificity, with up to 50 % of the trans-isomer being observed [30,32]. One explanation for this higher level of trans addition of protons is a rearrangement of a semi-reduced intermediate species bound to FeMo-cofactor during catalysis.…”

Section: Discussionmentioning

confidence: 99%

Alkyne substrate interaction within the nitrogenase MoFe protein

Santos

Mayer

Barney

et al. 2007

Journal of Inorganic Biochemistry

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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.

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Azotobacter vinelandii Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on the Catalyzed Reduction of Acetylene and Ethylene

Cited by 55 publications

References 46 publications

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Photolysis of Hi‐CO Nitrogenase – Observation of a Plethora of Distinct CO Species Using Infrared Spectroscopy

Alkyne substrate interaction within the nitrogenase MoFe protein

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