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 [2Fe-2S] ferredoxin from vegetative cells of the cyanobacterium Anabaena 7120, a prototypical plant-type ferredoxin, is described. The novel metal-substituted analog was obtained through constitution of the apoprotein with Ga(III) and sulfide anion in argon-saturated aqueous buffer. The replacement product migrated identically with the diferric protein on a 15% native PAGE gel and contained 2 atom equiv of gallium and sulfide per mole of protein, according to inductively coupled plasma and colorimetric analysis, respectively. The EXAFS spectrum of the gallium−sulfide constituted protein indicated the presence of a [2Ga-2S] cluster 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 1 H NMR spectra of the [2Fe-2S] ferredoxin appeared at chemical shifts essentially equivalent to those arising from analogous nuclei in the spectra of the [2Ga-2S] protein. Spectra of the gallium derivative, however, also possessed additional resonances; for example, 19 additional H α −H N cross peaks were observed in the fingerprint region of the DQF-COSY spectrum of the [2Ga-2S] protein taken in H 2 O 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 hyperfine-shifted or broadened beyond detectability in the iron−sulfur protein spectra. Preliminary analysis of the NMR spectra indicated that the structural features of the gallium−sulfide constituted protein possessed many similarities to those of the [2Fe-2S] protein in the crystalline state. Gallium-substituted analogs of iron−sulfur proteins should find utility in characterization of the structural and functional features of the ferric forms of the native iron−sulfur proteins using conventional NMR techniques.