Characterization of a supercluster and superspin model protein

The putative [6Fe-6S] prismane cluster in the 6-Fe/S-containing protein from Desulfovibrio vulgaris, strain Hildenborough, has been enriched to 80% in "Fe, and has been characterized in detail by S, X-, Pand Q-band EPR spectroscopy, parallel-mode EPR spectroscopy and high-resolution 57Fe Mossbauer spectroscopy. In EPR-monitored redox-equilibrium titrations, the cluster is found to be capable of three one-electron transitions with midpoint potentials at pH 7.5 of +285, +5 and 165 mV. As the fully reduced protein is assumed to carry the [6Fe-6SI3+ cluster, by spectroscopic analogy to prismane model compounds, four valency states are identified in the titration experiments : [6Fe-6SI3+, [6Fe-6SI4+, [6Fe-6SIs +, [6Fe-6SI6+. The fully oxidized 6 + state appears to be diamagnetic at low temperature. The prismane protein is aerobically isolated predominantly in the one-electronreduced 5+ state. In this intermediate state, the cluster exists in two magnetic forms: 10% is lowspin S = 112; the remainder has an unusually high spin S = 912. The S = 112 EPR spectrum is significantly broadened by ligand (2.3 mT) and ' 7Fe (3.0 mT) hyperfine interaction, consistent with a delocalization of the unpaired electron over 6Fe and indicative of at least some nitrogen ligation. At 35 GHz, the g tensor is determined as 1.971, 1.951 and 1.898. EPR signals from the S=9 /2 multiplet have their maximal amplitude at a temperature of 12 K due to the axial zero-field splitting being negative, D w -0.86 cm-l. Effective g = 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of I E/D I = 0.061. A second component has g = 9.7,8.1 and 6.65 and I E/D I = 0.108. When the protein is reduced to the 4+ intermediate state, the cluster is silent in normal-mode EPR. An asymmetric feature with effective g x 16 is observed in parallel-mode EPR from an integer spin system with, presumably, S = 4. The fully reduced 3 + state consists of a mixture of two S = 112 ground state. The g tensor of the major component is 2.010, 1.825 and 1.32; the minor component has g = 1.941 and 1.79, with the third value undetermined. The sharp line at g = 2.010 exhibits significant convoluted hyperfine broadening from ligands (2.1 mT) and from 57Fe (4.6 mT). Zerofield high-temperature Mossbauer spectra of the protein, isolated in the 5 + state, quantitatively account for the 0.8 fractional enrichment in 57Fe, as determined with inductively coupled plasma mass spectrometry. The six irons are not equivalent; the six quadrupole pairs are in a 2: 1 pattern. Upon reduction to the 3 + state, the spectra change shape dramatically with indication of localized valencies. Four of the six irons appear to be relatively unaffected, while the remaining two exhibit a considerable increase in quadrupole splitting and an increase in the isomer shift, each consistent with a full charge reduction. From temperature and field-dependent Mossbauer studies on the 5 + and 3 f states, it is concluded that all six irons are paramagnetic and part of the same spin system. A mixed-ligand prismane model is proposed in which four Fe form an electron-delocalized core, flanked on opposite sites by two Fe of distinctly more ionic character, as they are coordinated by nitrogen. In the corresponding vector-coupling model for the S = 912 state, the two ionic ferric ions couple ferromagnetically through the delocalized core structure. With the characterization of this model protein, a frame of reference is provided for the spectroscopic study of more complex Fe/S enzymes.