Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.

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Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange

Biochemical Journal ◽

10.1042/bj2770735 ◽

1991 ◽

Vol 277 (3) ◽

pp. 735-741 ◽

Cited By ~ 10

Author(s):

R N F Thorneley ◽

G A Ashby ◽

C Julius ◽

J L Hunter ◽

M R Webb

Keyword(s):

Klebsiella Pneumoniae ◽

Atp Hydrolysis ◽

Oxygen Exchange ◽

Cleavage Reaction ◽

Protein Ratio ◽

Mofe Protein ◽

Fe Protein ◽

Water Oxygen ◽

Atpase Reaction ◽

Protein Dissociation

The steady-state kinetics of reductant-independent ATP hydrolysis by Klebsiella pneumoniae nitrogenase at 23 degrees C at pH 7.4 were determined as a function of component protein ratio (optimal at an oxidized Fe protein/MoFe protein ratio of 3:1) and MgATP concentration (Km 400 microM). Competitive inhibition was observed for MgADP (Ki 145 microM), [beta gamma-methylene]ATP (Mgp[CH2]ppA) (Ki 115 microM), [beta gamma-monofluoromethylene]ATP (Mgp[CHF]ppA) (Ki 53 microM) and [beta gamma-difluoromethylene]ATP (Mgp[CF2]ppA) (Ki 160 microM). The tighter binding of MgADP to free oxidized Fe protein (KD less than 10 microM) than to the oxidized Fe protein-MoFe protein complex (Ki 145 microM) is proposed as the driving force that induces rate-limiting protein dissociation in the catalytic cycle of nitrogenase. The reversible nature of the reductant-independent MgATP-cleavage reaction was demonstrated by an MgADP-induced enhancement of the rate of the phosphate/water oxygen exchange reaction with 18O-labelled phosphate ion. This enhancement, like the reductant-independent ATPase reaction, only occurred with the complex formed by oxidized Fe protein and MoFe protein and not with the individual proteins. The results are discussed in terms of the mechanism of ATP hydrolysis by nitrogenase and other systems involving protein-protein interactions.

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Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur ‘P’ clusters

Biochemical Journal ◽

10.1042/bj2910709 ◽

1993 ◽

Vol 291 (3) ◽

pp. 709-711 ◽

Cited By ~ 6

Author(s):

R W Miller ◽

B E Smith ◽

R R Eady

Keyword(s):

Gel Filtration ◽

Good Evidence ◽

Published Data ◽

Midpoint Potential ◽

Mofe Protein ◽

Fe Protein ◽

The Press ◽

Pattern Characteristic ◽

Iron Molybdenum Cofactor ◽

Hydrolysis Of

Hydrolysis of MgATP to MgADP is essential for nitrogenase action. There is good evidence for binding of both nucleotides to the Fe protein of nitrogenase, but data indicating their binding to the MoFe protein have been controversial [see Miller and Eady (1989) Biochem. J. 263, 725-729]. The binding of MgADP to the MoFe protein of nitrogenase of Klebsiella pneumoniae was investigated by non-equilibrium gel-filtration column methods. No binding of MgADP to the dithionite-reduced protein could be detected. Treatment of the MoFe protein with phenosafranine [midpoint potential (Em) -270 mV] did not affect the activity, and oxidized the ‘P’ clusters but not the iron-molybdenum cofactor (FeMoco) centres. This oxidized species bound 3.9 mol of MgADP with a binding pattern characteristic of low rates of ligand dissociation. These observations suggest that the variability in published data on nucleotide binding to the MoFe protein is related to poor control of the protein oxidation level. Our data, coupled with the observation that ‘P’ clusters become oxidized during reduction of N2 [Lowe, Fisher and Thorneley (1993) Biochem. J., in the press], led us to propose that the ADP binding sites are transiently filled during enzyme turnover by hydrolysis of ATP originally bound to the Fe protein, and that hydrolysis occurs on a bridging site on the MoFe-Fe-protein complex.

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Maximal Efficiency of Coupling between ATP Hydrolysis and Translocation of Polypeptides Mediated by SecB Requires Two Protomers of SecA

Journal of Bacteriology ◽

10.1128/jb.01321-08 ◽

2008 ◽

Vol 191 (3) ◽

pp. 978-984 ◽

Cited By ~ 20

Author(s):

Chunfeng Mao ◽

Simon J. S. Hardy ◽

Linda L. Randall

Keyword(s):

Escherichia Coli ◽

Ternary Complex ◽

Atp Hydrolysis ◽

Protein Export ◽

Maximal Efficiency ◽

Low Efficiency ◽

Hydrolysis Of ◽

Low Activity

ABSTRACT SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.

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Covalent modification of nitrogenase MoFe protein by ADP

Biochemical Journal ◽

10.1042/bj3220737 ◽

1997 ◽

Vol 322 (3) ◽

pp. 737-744 ◽

Cited By ~ 3

Author(s):

Richard W. MILLER ◽

Robert R. EADY ◽

Carol GORMAL ◽

Shirley A. FAIRHURST ◽

Barry E. SMITH

Keyword(s):

Time Course ◽

Atp Hydrolysis ◽

Adenine Nucleotides ◽

Covalent Modification ◽

Mofe Protein ◽

Adenine Ring ◽

Fe Protein ◽

Lysine Residues ◽

Catalytically Active ◽

Nucleotide Interactions

MgADP- reacted with the nitrogenase molybdenum–iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad 31P NMR resonance at -1 p.p.m. lacking phosphorus J coupling. The adenine ring of [8-14C]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. Extended dialysis of Kp1 against MgADP- resulted in further AMP binding at the protein surface. ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites. ATP did not replace ADP. The time course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential. Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV. Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1. The adenine ring of ADP exchanged with adenine of MgATP2- during reductant-limited turnover of nitrogenase under N2, indicating reversibility of ATP hydrolysis at 15 °C. [32P]Pi exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1. 32P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalaldehyde (OPT). Preincubation with MgADP- protected against OPT inactivation. Two potentially reactive lysine residues on the α chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase Fe protein are proposed as sites of OPT and nucleotide binding. Azotobacter vinelandiiMoFe protein (Av1) also formed an AMP adduct but Kp2 did not. Catalase did not interact with ADP. The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterpart in known protein–nucleotide interactions.

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Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68922-2 ◽

1981 ◽

Vol 256 (16) ◽

pp. 8845-8849 ◽

Cited By ~ 8

Author(s):

G.M. Weinstock ◽

K. McEntee ◽

I.R. Lehman

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Kinetic Analysis ◽

Atp Hydrolysis ◽

Reca Protein ◽

Steady State Kinetic ◽

Nucleoside Triphosphates ◽

Hydrolysis Of ◽

State Kinetic

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2P194 Single molecule observation of ATP hydrolysis of Myosin V

Seibutsu Butsuri ◽

10.2142/biophys.45.s168_2 ◽

2005 ◽

Vol 45 (supplement) ◽

pp. S168

Author(s):

T. Komori ◽

S. Nishikawa ◽

T. Ariga ◽

A.H. Iwane ◽

H. Yamakawa ◽

...

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Myosin V ◽

Hydrolysis Of

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The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

Biochemical Journal ◽

10.1042/bj1200015 ◽

1970 ◽

Vol 120 (1) ◽

pp. 15-24 ◽

Cited By ~ 18

Author(s):

P. S. G. Goldfarb ◽

R. Rodnight

Keyword(s):

Potassium Chloride ◽

Magnesium Chloride ◽

Adenosine Triphosphatase ◽

Time Course ◽

Atp Hydrolysis ◽

Protein Ratio ◽

Low Concentrations ◽

Hydrolysis Of ◽

Emission Flame

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.

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Electron transfer and half-reactivity in nitrogenase

Biochemical Society Transactions ◽

10.1042/bst0390201 ◽

2011 ◽

Vol 39 (1) ◽

pp. 201-206 ◽

Cited By ~ 2

Author(s):

Thomas A. Clarke ◽

Shirley Fairhurst ◽

David J. Lowe ◽

Nicholas J. Watmough ◽

Robert R. Eady

Keyword(s):

Electron Transfer ◽

Protein Molecule ◽

Stopped Flow ◽

Protein Binding Sites ◽

Molybdenum Iron Protein ◽

Iron Protein ◽

Mofe Protein ◽

Fe Protein ◽

Rapid Quench ◽

Important Enzyme

Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum–iron protein (MoFe protein) and a protein containing an [Fe4–S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

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Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽

10.3390/antibiotics10121456 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1456

Author(s):

Amaravadhi Harikishore ◽

Chui-Fann Wong ◽

Priya Ragunathan ◽

Dennis Litty ◽

Volker Müller ◽

...

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Membrane Vesicles ◽

Α Subunit ◽

Rotational States ◽

C Terminus ◽

Inverted Membrane Vesicles ◽

Hydrolysis Of ◽

Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

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