Formation and characterization of a transition state complex of <i>Azotobacter vinelandii</i> nitrogenase

Duyvis, M.G.; Wassink, Hans; Haaker, H.

doi:10.1016/0014-5793(96)00019-1

Cited by 68 publications

(65 citation statements)

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“…This study would not be sensitive to electronic changes at Fe, nor to slight structural changes at the Fe sites, such as the binding of a light atom that would not lead to changes in the interatomic MoÐFe distances or order in the cluster. Therefore, the conclusions presented here, while in agreement with previous structural, biochemical and EPR results (Duyvis et al, 1996;Schindelin et al, 1997;Spee et al, 1998) and the Thorneley±Lowe kinetic scheme, which does not predict activity at the FeMoco center until several cycles of Fe protein complexation have occurred (Thorneley & Lowe, 1985), do not completely eliminate the possibility that some minor change has occurred at Fe in FeMoco in the ADPÁAlF 4 À -stabilized complex. A lack of change in the Mo environment of FeMoco in the complex strongly implies that minimal change has occurred at FeMoco and, therefore, raises questions as to how the MoFe protein and its metalloclusters are affected by the cycle of complex formation and electron transfer.…”

Section: Nitrogenase Complex Electronic Resultssupporting

confidence: 82%

“…To better understand the roles of the component protein interaction and nucleotide hydrolysis in electron transfer and substrate reduction, the ADPÁAlF 4 À -stabilized Fe protein± MoFe protein complex has been studied (Duyvis et al, 1996;Renner & Howard, 1996). Aluminium¯uoride is widely used as a -phosphate mimic to study the transition state of nucleotide hydrolysis (Wittinghofer, 1997).…”

Section: Introductionmentioning

confidence: 99%

“…Because ATP hydrolysis only occurs in the complex between the Fe protein and the MoFe protein, treatment of the component proteins together with MgADP and AlF 4 À stabilizes this complex. The Azotobacter vinelandii stabilized nitrogenase complex is not active in acetylene reduction or hydrogen evolution, which presumably indicates that FeMoco is not in a state that mimics turnover conditions (Duyvis et al, 1996;Renner & Howard, 1996;Yousafzai & Eady, 1999). Obligate hydrogen evolution, a process requiring two electrons, has been shown to occur, however, in the Klebsiella pneumoniae ADPÁAlF 4 À -stabilized nitrogenase complex and in a tight complex of Azotobacter vinelandii MoFe protein with Clostridium pasteurianum Fe protein (Clarke et al, 2000;Miller et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

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MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF₄⁻-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

Corbett

Tezcan

Einsle

et al. 2004

J Synchrotron Radiat

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The utility of using X-ray absorption spectroscopy (XAS) to study metalloproteins and, speci®cally, the enzyme complex nitrogenase, is highlighted by this study comparing both the structural and Mo-localized electronic features of the iron±molybdenum cofactor (FeMoco) in isolated MoFe protein and in the ADPÁAlF 4 À -stabilized complex of the MoFe protein with the Fe protein. No major differences are found at Mo between the two protein forms. The excellent quality of the data at both the Mo K and L edges will provide a baseline for analysis of other intermediates in the nitrogenase cycle. A new capability to delineate various contributions in the resting state of FeMoco is being pursued through polarized single-crystal XAS. The initial results point to the feasibility of using this technique for the analysis of scattering from the as yet unidenti®ed atom at the center of FeMoco.

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Section: Nitrogenase Complex Electronic Resultssupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF₄⁻-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

Corbett

Tezcan

Einsle

et al. 2004

J Synchrotron Radiat

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“…g x ϭ 2.115, g y ϭ 1.935, and g z ϭ 1.917 for [4Fe-4S]-I and g x ϭ 2.14, g y ϭ 2.008, and g z ϭ 1.89 for [4Fe-4S]-II (Fig. 3B, a, Ϫ can be considered to represent the trigonal bipyramidal geometry of the ␥-phosphate in the transition state undergoing nucleophilic attack by a water molecule (20,40). However, in the MgADP⅐AlF 4 Ϫ complex of DPOR the catalytic cycle was trapped before electron transfer occurred.…”

Section: Epr Analyses Of [4fe-4s] Clusters In the Ternary Dpor Complementioning

confidence: 99%

Biosynthesis of (Bacterio)chlorophylls

Bröcker

Wätzlich

Saggu

et al. 2010

Journal of Biological Chemistry

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Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL 2 subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB) 2 , which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB) 2 (ChlL 2 ) 2 complex after incubation with the nonhydrolyzable ATP analogs adenosine 5-(␥-thio)triphosphate, adenosine 5-(␤,␥-imido)triphosphate, or MgADP in combination with AlF 4 ؊ . Additionally, a mutant ChlL 2 protein, with a deleted Leu 153 in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL 2 , indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL 2 after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced. Reduction of protochlorophyllide a (Pchlide)2 to chlorophyllide a (Chlide) is a central step in the biosynthesis of chlorophyll and bacteriochlorophyll (1). Two evolutionarily unrelated enzymes are capable of catalyzing the stereospecific two-electron reduction of the C17-C18 double bond of Pchlide (Fig. 1A) (2-4). Monomeric, light-dependent Pchlide oxidoreductase (POR; NADPH Pchlide oxidoreductase, EC 1.3.1.33) drives the NADPH-dependent reduction (5-7) of Pchlide bound in the active site via absorption of light energy. This light dependence of POR catalysis prevents angiosperms from synthesizing chlorophyll in the dark (8, 9). Anoxygenicphotosynthetic bacteria make use of a different ATP-dependent Pchlide reducing system, which is termed the light-independent, dark operative Pchlide oxidoreductase (DPOR), whereas gymnosperms, mosses, ferns, algae, and cyanobacteria utilize both POR and DPOR (3). In chlorophyll-synthesizing organisms DPOR is encoded by the chlN, chlB, and chlL genes (10 -12). The corresponding genes for bacteriochlorophyllsynthesizing organisms have been termed bchN, bchB, and bchL (10, 13). DPOR subunits ChlN, ChlB, and ChlL share significant amino acid sequence homologies with nitrogenase subunits NifD, NifK, and NifH, respectively (12,14).In

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“…If the similarity between the Fe protein of nitrogenase and the molecular switch proteins is more than a structural one, it could be expected that nucleotide binding and hydrolysis are used by nitrogenase to switch between different conformations (1). Recently we (11) and Renner and Howard (12) have independently demonstrated that MgADP plus aluminum fluoride inhibits nitrogenase by stabilizing a complex between both nitrogenase proteins, suggesting a mode of action similar to that observed for molecular switch proteins (11)(12)(13). However, limited data are available that show that conformational changes occur in the nitrogenase complex during turnover.…”

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confidence: 99%

Pre-steady-state Kinetics of Nitrogenase from Azotobacter vinelandii

Duyvis

Wassink

Haaker

1996

Journal of Biological Chemistry

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The pre-steady-state electron transfer reactions of nitrogenase from Azotobacter vinelandii have been studied by stopped-flow spectrophotometry.With reduced nitrogenase proteins after the initial absorbance increase at 430 nm (which is associated with electron transfer from the Fe protein to the MoFe protein and is complete in 50 ms) the absorbance decreases, which, dependent on the ratio [Av2]/[Av1], is followed by an increase of the absorbance. The mixing of reductantfree nitrogenase proteins with MgATP leads after 20 ms to a decrease of the absorbance, which could be fitted (from 0.05 to 1 s) with a single exponential decay with a rate constant k obs ‫؍‬ 6.6 ؎ 0.8 s It is proposed that in the presence and absence of reductant, the observed absorbance decrease at 430 nm of nitrogenase is caused by a change of the conformation of the nitrogenase complex, as a consequence of hydrolysis of MgATP.

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Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

Cited by 68 publications

References 22 publications

MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF₄⁻-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF₄⁻-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

Biosynthesis of (Bacterio)chlorophylls

Pre-steady-state Kinetics of Nitrogenase from Azotobacter vinelandii

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Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

Cited by 68 publications

References 22 publications

MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF4−-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF4−-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

Biosynthesis of (Bacterio)chlorophylls

Pre-steady-state Kinetics of Nitrogenase from Azotobacter vinelandii

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MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF₄⁻-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

MoK- andL-edge X-ray absorption spectroscopic study of the ADP·AlF₄⁻-stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal