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

摘要

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.