A pure S=3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin

The properties of the [4Fe-4S](2+/+) cluster in wild-type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose alpha-C is less than 4 Angstrom from one of the cluster iron atoms by a tyrosine residue, Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38-mV decrease in the midpoint potential of the [4Fe-4S](2+,+) couple and has a marked effect on the ground state properties of the reduced cluster, The mixed spin [4Fe-4S](+) cluster in the wild-type protein, 80% S=3/2 (E/D=0.22, D=+3.3 cm(-1)) and 20% S=1/2 (g=2.10, 1.87, 1.80), is converted into a homogeneous S=3/2 (E/D=0.30, D=-0.7 cm(-1)) form in the A33Y variant, As the first example of a pure cm S=3/2 [4Fe-4S](+) cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe-4S](+) clusters. (C) 1999 Federation of European Biochemical Societies.