PREPARATION AND CHARACTERIZATION OF [2GA-2S] ANABAENA-7120 FERREDOXIN, THE FIRST GALLIUM-SULFUR CLUSTER-CONTAINING PROTEIN

The preparation and characterization of the [2Ga-2S] analog of the [2F e-2S] ferredoxin from vegetative cells of the cyanobacterium Anabaena 7120, a prototypical plant-type ferredoxin, is described. The novel me tal-substituted analog was obtained through constitution of the apopro tein with Ga(III) and sulfide anion in argon-saturated aqueous buffer. The replacement product migrated identically with the diferric protei n on a 15% native PAGE gel and contained 2 atom equiv of gallium and s ulfide per mole of protein, according to inductively coupled plasma an d colorimetric analysis, respectively. The EXAFS spectrum of the galli um-sulfide constituted protein indicated the presence of a [2Ga-2S] cl uster with structural features similar to those of [2Fe-2S] clusters. Cross peaks from nuclei that were not affected by the paramagnetism of the iron-sulfur cluster in two-dimensional H-1 NMR spectra of the [2F e-2S] ferredoxin appeared at chemical shifts essentially equivalent to those arising from analogous nuclei in the spectra of the [2Ga-2S] pr otein. Spectra of the gallium derivative, however, also possessed addi tional resonances; for example, 19 additional H-alpha-H-N cross peaks were observed in the fingerprint region of the DQF-COSY spectrum of th e [2Ga-2S] protein taken in H2O at 298 K. Sequential assignment of the resonances confirmed that the additional cross peaks originated from amino acids in the vicinity of the metal-sulfur cluster, which were hy perfine-shifted or broadened beyond detectability in the iron-sulfur p rotein spectra. Preliminary analysis of the NMR spectra indicated that the structural features of the gallium-sulfide constituted protein po ssessed many similarities to those of the [2Fe-2S] protein in the crys talline state. Gallium-substituted analogs of iron-sulfur proteins sho uld find utility in characterization of the structural and functional features of the ferric forms of the native iron-sulfur proteins using conventional NMR techniques.