A pure S=3/2 [Fe 4S 4] + cluster in the A33Y variant of Pyrococcus furiosus ferredoxin

Abstract

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 α-C is less than 4 Å 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 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.