Multi‐frequency EPR and high‐resolution Mössbauer spectroscopy of a putative [6Fe‐6S] prismane‐cluster‐containing protein from Desulfovibrio vulgaris (Hildenborough)
Authors: PIERIK Antonio J. , HAGEN Wilfred R. , DUNHAM W. Richard , SANDS Richard H. 
Published: March 2005

The putative [6Fe‐6S] prismane cluster in the 6‐Fe/S‐containing protein from Desulfovibrio vulgaris, strain Hildenborough, has been enriched to 80% in 57Fe, and has been characterized in detail by S‐, X‐, P‐ and Q‐band EPR spectroscopy, parallel‐mode EPR spectroscopy and high‐resolution 57Fe Mössbauer 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 lowspin 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 57Fe (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∼ ‐0.86 cm−1. Effective g= 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of ∣E/D∣= 0.061. 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∼ 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 57Fe (4.6 mT). Zerofield high‐temperature Mössbauer 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 appears 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 Mössbauer 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.