A CONTINUOUS, SPECTROPHOTOMETRIC ACTIVITY ASSAY FOR NITROGENASE USING THE REDUCTANT TITANIUM(III) CITRATE

A continuous, spectrophotometric assay for determining electron transfer rates through nitrogenase during substrate reduction reactions was developed. The assay takes advantage of the facts that TI(III) citrate can serve as a reductant for nitrogenase-catalyzed reduction reactions and that oxidation of Ti(III) citrate to Ti(IV) citrate results in a dramatic change in its absorption spectrum. Ti(III) citrate supported nitrogenase-catalyzed substrate (e.g., H+ or acetylene) reduction reactions at about the same rate as that supported by the reductant dithionite (S2O42-), In addition, Ti(III) citrate had an absorption maximum centered at 325 nm, while oxidized Ti(IV) citrate had a much lower absorption in this wavelength region. An absorption coefficient for Ti(III) citrate of 0.73 mM(-1).cm(-1) at 340 nm was determined by titration with redox dyes with known absorption coefficients. Using this experimentally determined absorption coefficient, we developed an assay that provides a convenient way to determine electron transfer rates through nitrogenase in real time by spectrophotometrically following the oxidation of Ti(III) citrate to Ti(IV) citrate. Average electron transfer rates of 3749 +/- 218 nmol of electrons transferred.min(-1).mg iron protein(-1) for H+ reduction were determined using this assay which are directly comparable to the rates calculated from fixed time point, gas chromatographic assays of H-2 formation. The utility of the TI(III) citrate assay for nitrogenase is discussed and demonstrated using the nitrogenase inhibitors MgADP, CN-, and NO. (C) 1994 Academic Press,Inc.