A reinvestigation of the pre‐steady‐state ATPase activity of the nitrogenase from <i>Azotobacter vinelandii</i>

Mensink, Richard E.; Wassink, Hans; Haaker, H.

doi:10.1111/j.1432-1033.1992.tb17185.x

Cited by 20 publications

(21 citation statements)

References 25 publications

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“…The current paper supplements the preceding communication [9] in which it was shown that the MgATPinduced H + production is much slower than electron transfer. Combined with the results of this paper, a model will be discussed in which the binding of MgATP changes the confor-mation of the protein complex which triggers electron transfer from Fe protein to MoFe protein.…”

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confidence: 64%

Temperature effects on the MgATP‐induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii

Mensink

Haaker

1992

European Journal of Biochemistry

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The temperature dependence of the pre-steady-state MgATP-dependent electron transfer from the MoFe protein to the Fe protein of the nitrogenase from Azotohacter vinelandii has been investigated between 6°C and 31 "C by stopped-flow spectrophotometry. Below 1 4 T , the data are consistent with a model in which interaction of MgATP with nitrogenase is fast and irreversible, and is followed by reversible electron transfer. From the extent and from the rate of the absorbance change, the rate constants for electron transfer from Fe protein to MoFe protein and of the reverse reaction were calculated. The direct rate constant increases with temperature (6-14°C) from about 1 s-l to about 26 s-'. The rate constant for the reverse reaction was found to be approximately 4 s -l and invariant with the reaction temperature. Analysis of the data obtained in the temperature range between 6°C and 12°C within the framework of the transition-state theory show that electron transfer from the Fe protein to the MoFe protein occurs via a highly disordered transition state with activation parameters AH": = 289 kJ . mol-' and A S ' ; = 792 J . K -l . mol-'. The Eyring plot of the stoppedflow data displays an inflection point around 14°C. From the stopped-flow data obtained between 18°C and 27°C the activation parameters AH": and AS" for the reduction of the MoFe protein by Fe protein are calculaled to be 90 kJ . mol-' and 99 J . K -' . mol-' respectively. A second inflection point in the Eyring plot could exist around 28°C.

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confidence: 64%

Temperature effects on the MgATP‐induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii

Mensink

Haaker

1992

European Journal of Biochemistry

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“…In all cases, the Cp2 concentration was 100 M, the Av1 concentration was 75 M, the buffer was 100 mM HEPES, pH 7.4, with 2 mM dithionite, and the samples were incubated for 1 h prior to freezing in liquid nitrogen. All EPR spectra were recorded at 12 K, with a 9.64-GHz microwave frequency, a 6.36-mW microwave power, a 100-kHz modulation frequency, and a modulation amplitude of 5.028 G. mary electron transfer from Cp2 to Av1 in the presence of MgATP represents more than a 10 4 -fold increase from the nucleotide free condition and approaches that observed for the MgATP-dependent rate constants found for the homologous A. vinelandii nitrogenase complex (42).…”

Section: Fig 2 Electron Transfer From the Cp2 [4fe-4s] Cluster To Av1mentioning

confidence: 65%

Evidence That MgATP Accelerates Primary Electron Transfer in aClostridium pasteurianum Fe Protein-Azotobacter vinelandii MoFe Protein Nitrogenase Tight Complex

Chan

Ryle

Seefeldt

1999

Journal of Biological Chemistry

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The nitrogenase catalytic cycle involves binding of the iron (Fe) protein to the molybdenum-iron (MoFe) protein, transfer of a single electron from the Fe protein to the MoFe protein concomitant with the hydrolysis of at least two MgATP molecules, followed by dissociation of the two proteins. Earlier studies found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum with the MoFe protein isolated from the bacterium Azotobacter vinelandii resulted in an inactive, nondissociating Fe protein-MoFe protein complex. In the present work, it is demonstrated that primary electron transfer occurs within this nitrogenase tight complex in the absence of MgATP (apparent first-order rate constant k ‫؍‬ 0.007 s ؊1) and that MgATP accelerates this electron transfer reaction by more than 10,000-fold to rates comparable to those observed within homologous nitrogenase complexes (k ‫؍‬ 100 s ؊1 Earlier studies (15)(16)(17) found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum (Cp2) with the MoFe protein isolated from the bacterium Azotobacter vinelandii (Av1) resulted in a nondissociating complex (Cp2⅐Av1) (see Equation 1).The association constant (K a ) for the Cp2⅐Av1 complex was determined to be approximately 10-fold higher than that for the homologous A. vinelandii nitrogenase complex and 100-fold higher than that for the homologous C. pasteurianum nitrogenase complex (15). In addition, this protein-protein complex could form with or without nucleotides (17). Although the Cp2⅐Av1 complex is inactive in all substrate reduction activities, it was later found (18) that this complex could still hydrolyze MgATP to MgADP ϩ P i at low rates (36-fold lower than the homologous protein complexes).In the present work, we present evidence that primary electron transfer, but not subsequent electron transfer reactions, occurs within the Cp2⅐Av1 tight complex. Primary electron transfer was found to occur without added nucleotides at low rates, but the addition of MgATP accelerated electron transfer by more than 10,000-fold to rates near those observed in the homologous protein complexes. How these results relate to our current understanding of the roles of MgATP in the nitrogenase mechanism is discussed. * This work was supported by National Science Foundation Grant MCB-9722937 (to L. C. S.) and by a Willard L. Eccles Foundation fellowship (to J. M. C). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed. n , oxidation state of the P cluster with all ferrous atoms; P ϩ , P cluster one-electron oxidized from the P n state; P 2ϩ , P cluster two-electron oxidized from the P n state; ⌬G, free energy change.

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“…landii ATCC strain 478 was grown and the nitrogenase component proteins were purified and assayed as described elsewhere (15). Specific activities of Av1 and Av2 were at least 8 and 2 mol⅐s Ϫ1 ⅐mol protein Ϫ1 of ethylene produced, respectively.…”

Section: Cell Growth and Isolation And Preparation Of Nitrogenase-a mentioning

confidence: 99%

“…Mensink and Haaker (14) concluded from their experiments on the temperature dependence of the extent and rate of MgATP-induced electron transfer that electron transfer occurs via a highly disordered transition state, which indicates a major conformational change. It was proposed that the binding of MgATP to the nitrogenase complex causes a change of the conformation of the complex that triggers electron transfer (14,15).…”

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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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A reinvestigation of the pre‐steady‐state ATPase activity of the nitrogenase from Azotobacter vinelandii

Cited by 20 publications

References 25 publications

Temperature effects on the MgATP‐induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii

Temperature effects on the MgATP‐induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii

Evidence That MgATP Accelerates Primary Electron Transfer in aClostridium pasteurianum Fe Protein-Azotobacter vinelandii MoFe Protein Nitrogenase Tight Complex

Pre-steady-state Kinetics of Nitrogenase from Azotobacter vinelandii

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