Evidence for a two-electron transfer using the all-ferrous Fe protein during nitrogenase catalysis

Andrew C. Nyborg, Joseph L. Johnson, Alexander Gunn, Gerald D. Watt

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Abstract

The nitrogenase-catalyzed H2 evolution and acetylene-reduction reactions using Ti(III) and dithionite (DT) as reductants were examined and compared under a variety of conditions. Ti(III) is known to make the all-ferrous Fe protein ([Fe4S4]0) and lowers the amount of ATP hydrolyzed during nitrogenase catalysis by approximately 2-fold. Here we further investigate this behavior and present results consistent with the Fe protein in the [Fe4S4]0 redox state transferring two electrons ([Fe4S4]2+/[Fe4S 4]0) per MoFe protein interaction using Ti(III) but transferring only one electron ([Fe4S4]2+/[Fe4S 4]1+) using DT. MoFe protein specific activity was measured as a function of Fe:MoFe protein ratio for both a one- and a two-electron transfer reaction, and nearly identical curves were obtained. However, Fe protein specific activity curves as a function of MoFe:Fe protein ratio showed two distinct reactivity patterns. With DT as reductant, typical MoFe inhibition curves were obtained for operation of the [Fe4S4]2+/ [Fe4S4]1+ redox couple, but with Ti(III) as reductant the [Fe4S4]2+/[Fe4S4 ]0 redox couple was functional and MoFe inhibition was not observed at high MoFe:Fe protein ratios. With Ti(III) as reductant, nitrogenase catalysis produced hyperbolic curves, yielding a Vmax for the Fe protein specific activity of about 3200 nmol of H2 min-1 mg-1 Fe protein, significantly higher than for reactions conducted with DT as reductant. Lag phase experiments (Hageman, R. V., and Burris, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2699-2702) were carried out at MoFe:Fe protein ratios of 100 and 300 using both DT and Ti(III). A lag phase was observed for DT but, with Ti(III) product formation, began immediately and remained linear for over 30 min. Activity measurements using Av-Cp heterologous crosses were examined using both DT and Ti(III) as reductants to compare the reactivity of the [Fe4S4]2+/[Fe4S4 ]1+ and [Fe4S4]2+/ [Fe4S4]0 redox couples and both were inactive. The results are discussed in terms of the Fe protein transferring two electrons per MoFe protein encounter using the [Fe4S4]2+/[Fe4S4 ]0 redox couple with Ti(III) as reductant.

Original languageEnglish (US)
Pages (from-to)39307-39312
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number50
DOIs
StatePublished - Dec 15 2000

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