MULTIFREQUENCY EPR AND HIGH-RESOLUTION MOSSBAUER-SPECTROSCOPY OF A PUTATIVE [6FE-6S] PRISMANE-CLUSTER-CONTAINING PROTEIN FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH) - 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-57, and has been characterized in detail by S-, X-, P- and Q-band EPR spectroscopy, parallel-mode EPR spectroscopy and high-resolution Fe-57 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-6S]3+ cluster, by spectroscopic analogy to prismane model compounds, four valency states are identified in the titration experiments: [6Fe-6S]3+,[6Fe-6S]4+, [6Fe-6S]5+, [6Fe-6S]6+. The fully oxidized 6+ state appears to be diamagnetic at low temperature. The prismane protein is aerobically isolated predominantly in the one-electron-reduced 5+ state. In this intermediate state, the cluster exists in two magnetic forms: 10% is low-spin S = 1/2; the remainder has an unusually high spin S = 9/2. The S = 1/2 EPR spectrum is significantly broadened by ligand (2.3 mT) and Fe-57 (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 almost-equal-to -0.86 cm-1. Effective g = 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of \E/D\ = 0.06 1. A second component has g = 9.7, 8.1 and 6.65 and \E/D\ = 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 almost-equal-to 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 = 1/2 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 Fe-57 (4.6 mT). Zero-field high-temperature Mossbauer spectra of the protein, isolated in the 5+ state, quantitatively account for the 0.8 fractional enrichment in Fe-57, 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+ 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 = 9/2 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.