Reduction of spinach and parsley ferredoxin FdI in the (FeFeIII)-Fe-III state with the 1,4,8,12-tetraazacyclopentadecane complex [Cr(15-aneN(4))(H2O)(2)](2+), here written as Cr(II)L, provides the first evidence for two 1-equiv steps yielding an (FeFeII)-Fe-II product. Rate constants (25 degrees C) for spinach FdI are 2760 and 660 M(-1) s(-1), respectively, at pH 7.5, I = 0.100 M (NaCI). An important observation is that the Cr(III)L generated in the first step remains attached to the (FeFeIII)-Fe-II product and perturbs the protein active site sufficiently to make the second stage possible. The second Cr(II)L reduction is of the "outer-sphere" type, and the Cr(III)L generated is not attached to the protein. Anaerobic reoxidation of the fully reduced protein with [Co(NH3)(6)](3+) is rapid and can be achieved with similar to 80% recovery of the (FeFeIII)-Fe-III oxidation state over 40 min. Air oxidation yields the Cr(III)L product (FeFeIII)-Fe-III... Cr(III)L (Fe:Cr = 2:1). With Anabaena variabilis only a one-step reduction is observed and there is no Cr(III)L attachment. From a comparison of amino acid sequences with spinach (and parsley) FdI, a likely point of Cr(III)L attachment is indicated. Comparisons are made with dithionite as reductant. In addition, square-wave voltammetry on spinach (FeFeIII)-Fe-III... Cr(III)L gives two reduction potentials -273 and -410 mV vs NHE. The different redox products have been characterized by EPR. Using H-1 NMR line-broadening techniques, evidence for Cr(III)L binding at a surface site close to Tyr-25/yr-82 is obtained. Also from investigations with redox-inactive [Cr(en)(3)](3+) as a competitive inhibitor for Cr(III)L reduction of spinach (FeFeIII)-Fe-III, Tyr-25/Tyr-82 is proposed as the site for Cr(III)L reduction. From an extension of studies to include reduction of (FeFeIII)-Fe-III... Cr(III)L with Cr(II)L, evidence is obtained for a second reaction site when that at Tyr-25/Tyr-82 is no longer available.