Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis

Johnson, Joseph L.; Nyborg, Andrew C.; Wilson, Phillip E.; Tolley, A M; Nordmeyer, Francis R.; Watt, Gerald D.

doi:10.1016/s0167-4838(00)00195-3

Cited by 17 publications

(26 citation statements)

References 28 publications

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“…At the saturation of Av1 by Av2, the specific activity of Av1 at pH 9.5 was estimated as 100 nmol min −1 mg −1 , or ~5% of the value at pH 7.8. As also observed at the optimal pH (29–31), the Av1 titration curve at pH 9.5 is not fully hyperbolic and has a “lag” at low concentrations of Av2 (CR < ~1). This is consistent with a similar process of interactions between the MoFe-protein and Fe-protein that is observed at the optimal pH and is maintained at the higher pH.…”

Section: Resultsmentioning

confidence: 55%

Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

et al. 2014

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Proton uptake accompanies the reduction of all known substrates by nitrogenase. As one consequence, higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high pH approach would appear problematic in view of the observation reported by Pham and Burgess (Biochemistry 32, 13725 (1993)) that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While normal, albeit low, substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially the enzyme is reversibly inhibited for substrate reduction at pH 9.5 yet in a second, slower process the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to native MoFe-protein yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by tight complex formation with Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism based transformation that may provide a new probe of the nitrogenase catalysis.

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Section: Resultsmentioning

confidence: 55%

Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

et al. 2014

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“…4 Dilution Effect-Thorneley and Lowe (13, 28) studied the dilution effect for nitrogenases from Klebsiella pneumoniae (Kp) and Azotobacter chroococcum (Ac) with DT as reductant and proposed that low protein concentrations prevent complex formation between the nitrogenase component proteins. The dilution effect was also recently examined with both Av and Cp and gave results comparable to both Kp and Ac (25). The dilution effect feature that is relevant to this study is the linear increase in product formation as a function of increasing MoFe protein concentration at a fixed Fe:MoFe protein ratio that extrapolates to a finite x intercept near 0.2-0.4 M. Fig.…”

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confidence: 62%

“…No more than six samples were removed from each reaction vial and corrections were made for the change in volume of the headspace gas due to the removal of consecutive 0.2-ml aliquots. Dilution experiments were carried out as described previously using 20 mM DT or 5.0 -7.0 mM Ti(III) as reductant (25).…”

mentioning

confidence: 99%

Evidence for a Two-Electron Transfer Using the All-Ferrous Fe Protein during Nitrogenase Catalysis

Nyborg¹,

Johnson²,

Gunn³

et al. 2000

Journal of Biological Chemistry

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“…The total Fe content of the nitrogenase complex depends on the ratio of Fe to MoFe protein (coded for by nifH and nifDK respectively) which has not to our knowledge been measured in Trichodesmium . Maximal specific activity for nitrogenase appears to occur at a ratio near 5 Fe:MoFe [62], [63], with structural analysis of the protein [20] and nifH/nifDK transcript products [64] indicating a range of ratios from 2–5. This corresponds to a possible Fe content of 38–50 mol Fe (mol nitrogenase complex) -1 , i.e.…”

Section: Resultsmentioning

confidence: 99%

Abundances of Iron-Binding Photosynthetic and Nitrogen-Fixing Proteins of Trichodesmium Both in Culture and In Situ from the North Atlantic

et al. 2012

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Marine cyanobacteria of the genus Trichodesmium occur throughout the oligotrophic tropical and subtropical oceans, where they can dominate the diazotrophic community in regions with high inputs of the trace metal iron (Fe). Iron is necessary for the functionality of enzymes involved in the processes of both photosynthesis and nitrogen fixation. We combined laboratory and field-based quantifications of the absolute concentrations of key enzymes involved in both photosynthesis and nitrogen fixation to determine how Trichodesmium allocates resources to these processes. We determined that protein level responses of Trichodesmium to iron-starvation involve down-regulation of the nitrogen fixation apparatus. In contrast, the photosynthetic apparatus is largely maintained, although re-arrangements do occur, including accumulation of the iron-stress-induced chlorophyll-binding protein IsiA. Data from natural populations of Trichodesmium spp. collected in the North Atlantic demonstrated a protein profile similar to iron-starved Trichodesmium in culture, suggestive of acclimation towards a minimal iron requirement even within an oceanic region receiving a high iron-flux. Estimates of cellular metabolic iron requirements are consistent with the availability of this trace metal playing a major role in restricting the biomass and activity of Trichodesmium throughout much of the subtropical ocean.

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Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis

Cited by 17 publications

References 28 publications

Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

Evidence for a Two-Electron Transfer Using the All-Ferrous Fe Protein during Nitrogenase Catalysis

Abundances of Iron-Binding Photosynthetic and Nitrogen-Fixing Proteins of Trichodesmium Both in Culture and In Situ from the North Atlantic

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