Potentiometric and electron nuclear double resonance properties of the two spin forms of the [4Fe-4S]+ cluster in the novel ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.
Pyrococcus furiosus ferredoxin contains a single [4Fe-4S] that exists in both S = 1/2 (20%) and S = 3/2 (80%) ground states in the reduced protein. We report here on the temperature-dependent potentiometric properties of the two spin forms, their stability, and on the structural features that differentiate them. The midpoint potential (Em) of the cluster in either spin state was determined at -365 mV (30 degrees C, pH 8.0). By rapidly freezing samples for EPR analyses, it was shown that the Em values of both spin states appear to change by -1.7 mV/degrees C over the range 20 degrees-80 degrees C, and by -6 mV/degrees C between 80 and 89 degrees C. The Em values and the relative amounts of the S = 1/2 and S = 3/2 forms of the cluster were unaffected by pH (6.8-10.5), even at 85 degrees C, and were unchanged by the presence of NaCl (1.0 M), sodium dodecyl sulfate (10%, w/v) or ethylene glycol (50%, v/v), even at 80 degrees C. The S = 1/2 form of the [4Fe-4S]+ cluster was found to exhibit a strongly coupled 1H ENDOR resonance (A = 22 MHz) that was exchangeable with the solvent. Such a large coupling has not been observed in any other iron-sulfur protein. Since a unique feature of this 4Fe-ferredoxin is that only 3 cysteinyl residues appear to be coordinated to the [4Fe-4S] cluster, the ENDOR data are consistent with an H2O molecule being a ligand to the unique Fe site. The S = 3/2 form of the [4Fe-4S]+ cluster exhibited a similar, strongly coupled 1H ENDOR resonance, but in this spin state it was not exchangeable with the solvent. This suggests that the [4Fe-4S]+ cluster exhibiting the S = 3/2, but not the S = 1/2 ground state, is "shielded" from the solvent, presumably by neighboring amino acid residues. In view of the pH dependence of the midpoint potential of the two spin states, the fourth ligand to the cluster and the source of the strongly coupled 1H ENDOR resonance is probably an OH- rather than H2O molecule.
Park
J B
JB
Department of Biochemistry, University of Georgia, Athens 30602.
Fan
C L
CL
Hoffman
B M
BM
Adams
M W
MW
D001105:Archaea / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011199:Potentiometry; D013696:Temperature
D005288:Ferredoxins