Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials when only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = 3/2 and 5/2 spin states of one reduced [4Fe-4S] cluster, No typical S = 1/2 EPR signals were observed. At lower potentials (less than - 500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = 1/2 low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = 1/2 and S = 5/2 EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >5/2. The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.