Evidence for a two-electron transfer using the all-ferrous Fe protein during nitrogenase catalysis.
The nitrogenase-catalyzed H(2) 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 ([Fe(4)S(4)](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 [Fe(4)S(4)](0) redox state transferring two electrons ([Fe(4)S(4)](2+)/[Fe(4)S(4)](0)) per MoFe protein interaction using Ti(III) but transferring only one electron ([Fe(4)S(4)](2+)/[Fe(4)S(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 [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple, but with Ti(III) as reductant the [Fe(4)S(4)](2+)/[Fe(4)S(4)](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 V(max) for the Fe protein specific activity of about 3200 nmol of H(2) 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 [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) and [Fe(4)S(4)](2+)/[Fe(4)S(4)](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 [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with Ti(III) as reductant.
Nyborg
A C
AC
Undergraduate Research Program and Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84604, USA.
Johnson
J L
JL
Gunn
A
A
Watt
G D
GD
D001395:Azotobacter / Q000378:metabolism; D002384:Catalysis; D019343:Citric Acid / Q000494:pharmacology; D004227:Dithionite / Q000494:pharmacology; D004579:Electron Transport; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008983:Molybdoferredoxin; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013997:Time Factors; D014025:Titanium / Q000494:pharmacology
D005296:Ferrous Compounds; D007506:Iron-Sulfur Proteins; D008983:Molybdoferredoxin; C419387:titanium citrate; D004227:Dithionite; D019343:Citric Acid; D006859:Hydrogen; D014025:Titanium; D009591:Nitrogenase