Studies on the structure of ferredoxin

Abstract

The preparation of apoferredoxin from spinach ferredoxin and ferredoxin of Clostridium pasteurianum with α,α′-dipyridyl without the addition of reducing agents is described. Reconstitution to form active ferredoxins can be achieved in high yields under various conditions. 2-Mercaptoethanol can be replaced by cysteamine or Cleland's Reagent. The reconstitution is also possible by using elemental sulfur, cysteamine, and iron(II) ammonium sulfate. The origin of labile sulfur in Clostridial ferredoxin has been investigated. The elemental analyses show conclusively that in ferredoxin there are 5–6 additional sulfurs besides the 8 sulfurs from the cysteine moieties, while apoferredoxin contains only the 8 sulfurs of the cysteine groups. A clostridial [35S] ferredoxin has been prepared by using Na235S in the resynthesis. The uptake of sulfide corresponded to 6 equivalents of sulfur per mole of ferredoxin. Treatment of the 35S-labelled clostridial ferredoxin yielded apoferredoxin essentially free of radioactivity. From all of our analytical data the most probable formulation for Clostridium pasteurianum includes 8 equivalents of cysteine, 6 equivalents of labile sulfur, and 6 equivalents of iron. The hydropersulfides of cysteamine and cysteine methylester were prepared in solution. Paramagnetic complexes with gav-values below 2 were prepared from the ferrous complex of cysteamine or cysteine methyl ester and hydropersulfides of cysteamine or cysteine methyl ester. The g -values of the persulfide complexes are 1.92, 1.94 and 2.00. The monomeric unit of these paramagnetic compounds is formulated as a disulfide-iron(II)-sulfide complex which is equivalent to a persulfide-thiolato-iron(II), and a sulfur complex of bis-thiolato-iron(II). If one considers the active center in plant ferredoxin as an analogous dimeric unit and the center in clostridial ferredoxin as an oligomeric unit, the labile sulfur may be represented in the form of S2° or disulfide anion S22− in oxidized ferredoxins or supersulfide anion S2⋅− in reduced ferredoxins. Structures are postulated for the active center of binuclear plant ferredoxins that are in agreement with the chemical and physical properties. These contain Fe–S2–Fe groups which are believed to be non-planar and exhibit stereochemistry similar to that found earlier by X-ray analysis for the binuclear complex (CO)3FeS2Fe(CO)3. The oxidized binuclear ferredoxins are described by the valence resonance hybrids Fe(II)S2°Fe(II) and Fe(III)S22−Fe(III), and the reduced paramagnetic form by Fe(II)S2⋅−-Fe(II) and Fe(II)S22−Fe(III). This seems to unite different theories concerning the cause for the characteristic ESR signal of ferredoxins. For polynuclear ferredoxins of the clostridial ferredoxin type an active center is suggested which contains units of binuclear ferredoxins in modified form. It appears that in the polynuclear active centers the formulation Fe(III)S22−Fe(III) participates more in the ground state. In the light of the chemical and physical properties it is proposed that in clostridial ferredoxin a multicenter three-dimensional iron-sulfur cluster exists rather than a pseudo-linear array.