The three-dimensional structure of the native 'putative prismane' protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72 Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12 Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N- terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ2-sulfido bridges, two μ2-oxo bridges, and a partially occupied, unidentified μ2 bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the 'hybrid' cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10K; no Fe··Fe distances beyond 3.1 Å could be identified. EPR, Mossbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]1+ cubane with S = 3/2; it is the first biological example of a 'spin-admixed' iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe(III) to three Fe(II) plus one Fe(III). The four iron ions are exchange coupled resulting in the system spins S = 0, 9/2, 0 (and 4), 1/2, respectively for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.