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 (\mathrm{Ti}() III) citrate can serve as a reductant for nitrogenase-catalyzed reduction reactions and that oxidation of (\mathrm{Ti}() III) citrate to (\mathrm{Ti}(\mathrm{IV})) citrate results in a dramatic change in its absorption spectrum. Ti(III) citrate supported nitrogenase-catalyzed substrate (e.g., (\mathrm{H}^{+})or acetylene) reduction reactions at about the same rate as that supported by the reductant dithionite (\left(\mathrm{S}{2} \mathrm{O}{4}{ }^{2-}\right)). In addition, Ti(III) citrate had an absorption maximum centered at (325 \mathrm{~nm}), while oxidized (T i(I V)) citrate had a much lower absorption in this wavelength region. An absorption coefficient for (\mathrm{Ti}) (III) citrate of (0.73 \mathrm{mM}^{-1} \cdot \mathrm{cm}^{-1}) at (340 \mathrm{~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 (\mathrm{Ti}(\mathrm{IV})) citrate. Average electron transfer rates of (3749 \pm 218 \mathrm{nmol}) of electrons transferred (\cdot \min ^{-1} \cdot \mathbf{m g}) iron protein ({ }^{-1}) for (\mathrm{H}^{+})reduction were determined using this assay which are directly comparable to the rates calculated from fixed time point, gas chromatographic assays of (\mathbf{H}_{2}) formation. The utility of the (\mathrm{Ti}(\mathrm{III})) citrate assay for nitrogenase is discussed and demonstrated using the nitrogenase inhibitors MgADP, (\mathrm{CN}^{-}), and NO. 1994 Academic Press, Inc.