Purification and biochemical characterization of a putative [6Fe‐6S] prismane‐cluster‐containing protein from Desulfovibrio vulgaris (Hildenborough)

Abstract

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 ± 0.4 Fe/polypeptide and 6.2 ± 0.7 S^2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S= 1/2 [6Fe-6S]³⁺ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI= 4.9). The ultraviolet/visible spectrum is relatively featureless (ɛ₂₈₀= 81 mM⁻¹· cm⁻¹, ɛ₄₀₀= 25 mM⁻¹· cm⁻¹, ɛ_400,red= 14 mM⁻¹· cm⁻¹). The shape of the protein is approximately globular (s_20,w= 4.18 S). The N-terminal amino acid sequence is MF^s/_cFQ^s/_c QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.