Formation, Properties, and Characterization of a Fully Reduced Fe(II)Fe(II) Form of Spinach (and Parsley) [2Fe-2S] Ferredoxin with the Macrocyclic Complex [Cr(15-aneN(4))(H(2)O)(2)](2+) as Reductant.

Abstract

Reduction of spinach and parsley ferredoxin FdI in the Fe(III)Fe(III) state with the 1,4,8,12-tetraazacyclopentadecane complex [Cr(15-aneN(4))(H(2)O)(2)](2+), here written as Cr(II)L, provides the first evidence for two 1-equiv steps yielding an Fe(II)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 (NaCl). An important observation is that the Cr(III)L generated in the first step remains attached to the Fe(II)Fe(III) 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(NH(3))(6)](3+) is rapid and can be achieved with approximately 80% recovery of the Fe(III)Fe(III) oxidation state over 40 min. Air oxidation yields the Cr(III)L product Fe(III)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 Fe(III)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 (1)H NMR line-broadening techniques, evidence for Cr(III)L binding at a surface site close to Tyr-25/Tyr-82 is obtained. Also from investigations with redox-inactive [Cr(en)(3)](3+) as a competitive inhibitor for Cr(II)L reduction of spinach Fe(III)Fe(III), Tyr-25/Tyr-82 is proposed as the site for Cr(II)L reduction. From an extension of studies to include reduction of Fe(III)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.