scholarly journals Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen?

1993 ◽  
Vol 292 (1) ◽  
pp. 93-98 ◽  
Author(s):  
D J Lowe ◽  
K Fisher ◽  
R N F Thorneley
Keyword(s):  
Steady State ◽  
Kinetic Model ◽  
Klebsiella Pneumoniae ◽  
The State ◽  
Protein Ratio ◽  
Mofe Protein ◽  
Absorbance Changes ◽  
Component Protein

The pre-steady-state absorbance changes that occur during the first 0.6 s of reaction of the nitrogenase of Klebsiella pneumoniae can be simulated by associating redox changes with the different states of the MoFe protein described by our published kinetic model for nitrogenase [Lowe and Thorneley (1984) Biochem. J. 224, 877-886]. When the substrate is changed, from H+ to C2H2 (acetylene) or N2, or the nitrogenase component protein ratio is altered, these pre-steady-state absorbance changes are affected in a manner that is quantitatively predicted by our model. The results, together with parallel e.p.r. studies, are interpreted as showing that the P-clusters become oxidized when the MoFe protein is in the state where bound N2 is irreversibly committed to being reduced and is protonated to the hydrazido(2-) level.

scholarly journals The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation

1984 ◽  
Vol 224 (3) ◽  
pp. 877-886 ◽  
Author(s):  
D J Lowe ◽  
R N Thorneley
Keyword(s):  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Necessary Condition ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Rapid Quench ◽  
Steady State Kinetic ◽  
H2 Formation

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.

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

1991 ◽  
Vol 277 (3) ◽  
pp. 735-741 ◽  
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.

scholarly journals Klebsiella pneumoniae nitrogenase. The pre-steady-state kinetics of MoFe-protein reduction and hydrogen evolution under conditions of limiting electron flux show that the rates of association with the Fe-protein and electron transfer are independent of the oxidation level of the MoFe-protein

1991 ◽  
Vol 279 (1) ◽  
pp. 81-85 ◽  
Author(s):  
K Fisher ◽  
D J Lowe ◽  
R N F Thorneley
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Electron Flux ◽  
High Electron ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Kinetics Of

The pre-steady-state kinetics of H2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23 degrees C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (kobs. = 140 s-1). These data support our previous assumption that the rate constants that define the ‘Fe-protein cycle’ are independent of the level of reduction of the MoFe-protein.

scholarly journals Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide

1990 ◽  
Vol 272 (3) ◽  
pp. 621-625 ◽  
Author(s):  
D J Lowe ◽  
K Fisher ◽  
R N F Thorneley
Keyword(s):  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Electron Flux ◽  
Lag Phase ◽  
High Electron ◽  
Mofe Protein ◽  
Fe Protein ◽  
C2h2 Reduction ◽  
Electron Reduction ◽  
State Concentration

The electron flux through the MoFe-protein of nitrogenase from Klebsiella pneumoniae determines the absolute and relative rates of 2H+ reduction to H2 and acetylene (C2H2) reduction to ethylene (C2H4) at saturating levels of reductant (Na2S2O4) and MgATP. High electron flux, induced by a high Fe-protein (Kp2)/MoFe protein (Kp1) ratio, favours C2H2 reduction. These data can be explained if ethylene, the two-electron reduction product of C2H2, is not released until three electrons have been transferred from Kp2 to Kp1. This explanation is also consistent with a pre-steady-state lag phase for C2H4 formation of 250 ms observed when functioning enzyme is quenched with acid. Electron flux through nitrogenase is inhibited by C2H2 at high protein concentrations. This is because the association rate between Kp1 and oxidized Kp2 is enhanced by C2H2, leading to an increased steady-state concentration of the inhibitory complex Kp2oxKp1C2H2. This effect is not relieved by CO. Thus CO and C2H2 (or C2H4) must be bound at the same time to distinct sites, presumably at Mo or Fe centres, on the enzyme.

scholarly journals The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution

1984 ◽  
Vol 224 (3) ◽  
pp. 895-901 ◽  
Author(s):  
D J Lowe ◽  
R N F Thorneley
Keyword(s):  
Klebsiella Pneumoniae ◽  
Rate Constants ◽  
Competitive Inhibitor ◽  
H2 Evolution ◽  
Protein Ratio ◽  
Mofe Protein ◽  
Fe Protein ◽  
Mutual Displacement ◽  
Kinetics Of

Kinetic data for Klebsiella pneumoniae nitrogenase were used to determine the values of nine of the 17 rate constants that define the scheme for nitrogenase action described by Lowe & Thorneley [(1984) Biochem. J. 224, 877-886]. Stopped-flow spectrophotometric monitoring of the MgATP-induced oxidation of the Fe protein (Kp2) by the MoFe protein (Kp1) was used to determine the rates of association (k+1) and dissociation (k-1) of reduced Kp2(MgATP)2 with Kp1. The dependences of the apparent KNm2 on Fe protein/MoFe protein ratio and H2 partial pressure were used to determine the mutual displacement rates of N2 and H2 (k+10, k-10, k+11 and k-11). These data also allowed the rate constants for H2 evolution from progressively more reduced forms of Kp1 to be determined (k+7, k+8 and k+9). A mechanism for N2-dependent catalysis of 1H2H formation from 2H2 that requires H2 to be a competitive inhibitor of N2 reduction is also presented.

scholarly journals Nitrogenase MoFe protein subunits from Klebsiella pneumoniae expressed in foreign hosts. Characteristics and interactions.

1987 ◽  
Vol 262 (18) ◽  
pp. 8814-8820 ◽  
Author(s):  
D Holland ◽  
A Zilberstein ◽  
D Govezensky ◽  
D Salomon ◽  
A Zamir
Keyword(s):  
Klebsiella Pneumoniae ◽  
Protein Subunits ◽  
Mofe Protein

scholarly journals Steady State Response of Linear Time Invariant Systems Modeledby Multibond Graphs

2021 ◽  
Vol 11 (4) ◽  
pp. 1717
Author(s):  
Gilberto Gonzalez Avalos ◽  
Noe Barrera Gallegos ◽  
Gerardo Ayala-Jaimes ◽  
Aaron Padilla Garcia
Keyword(s):  
Steady State ◽  
Linear Time ◽  
Direct Determination ◽  
The State ◽  
Steady State Response ◽  
State Variables ◽  
Linear Time Invariant ◽  
State Response ◽  
Time Invariant

The direct determination of the steady state response for linear time invariant (LTI) systems modeled by multibond graphs is presented. Firstly, a multiport junction structure of a multibond graph in an integral causality assignment (MBGI) to get the state space of the system is introduced. By assigning a derivative causality to the multiport storage elements, the multibond graph in a derivative causality (MBGD) is proposed. Based on this MBGD, a theorem to obtain the steady state response is presented. Two case studies to get the steady state of the state variables are applied. Both cases are modeled by multibond graphs, and the symbolic determination of the steady state is obtained. The simulation results using the 20-SIM software are numerically verified.

Serial analysis of renal blood flow by positron tomography with rubidium-82

1986 ◽  
Vol 251 (5) ◽  
pp. H1024-H1030
Author(s):  
N. Tamaki ◽  
C. A. Rabito ◽  
N. M. Alpert ◽  
T. Yasuda ◽  
J. A. Correia ◽  
...  
Keyword(s):  
Blood Flow ◽  
Steady State ◽  
Kinetic Model ◽  
Renal Blood Flow ◽  
Constant Infusion ◽  
Flow Reduction ◽  
Arterial Blood ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Rubidium 82

To determine whether renal blood flow can be measured by positron-emission tomography (PET) during constant infusion of rubidium-82 (82Rb) using a steady-state kinetic model, studies were performed in 10 dogs at control (n = 10), during mild flow reduction (n = 7), during severe flow reduction (n = 10), and after reperfusion of the kidney (n = 3). PET data were quantified to determine mean concentration of 82Rb (Ct) in each transverse section of the kidney. The arterial concentration (Ca) of 82Rb was measured by well counting of arterial blood samples during the equilibrium scan. 82Rb renal uptake (Ct/Ca) correlated nonlinearly with microsphere renal blood flow according to a steady-state kinetic model (r = 0.90). 82Rb estimated flow was 3.16 +/- 1.36 ml X min-1 X g-1 at control and 1.56 +/- 0.57 and 0.37 +/- 0.59 during mild and severe flow reductions, respectively. Microsphere determined flow was 2.89 +/- 0.77 ml X min-1 X g-1 at control, 1.58 +/- 0.42 at mild reduction, and 0.27 +/- 0.49 at severe reduction. In the occlusion and reperfusion model, the 82Rb estimated flow during occlusion was 0.21 +/- 0.15 ml X min-1 X g-1 and on reperfusion went up to 2.13 +/- 1.08. The contralateral kidney demonstrated reductions in the 82Rb estimated flow of 3.02 +/- 1.62 ml X min-1 X g-1 (63%) and 2.92 +/- 0.89 (61%) during mild and severe flow reductions, respectively. We conclude that PET with 82Rb permits serial quantitative assessment of renal flood flow.

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