INTERACTION OF TL+ AND CS+ WITH THE [FE3S4] CLUSTER OF PYROCOCCUS-FURIOSUS FERREDOXIN - INVESTIGATION BY RESONANCE RAMAN, MCD, EPR, AND ENDOR SPECTROSCOPY

The hyperthermophilic archaeon Pyrococcus furiosus contains a novel 4Fe ferredoxin in which one Fe ion lacks cysteinyl coordination. This unique Fe ion can be easily removed to yield protein containing a [Fe3S4](0) cluster. Under reducing conditions, this cluster can bind exogenous metal dications, M(2+) (e.g., Ni2+ and Zn2+), to yield [MFe(3)S(4)](+) clusters. In this work, we have investigated the affinity of the [Fe3S4](0,+) + cluster in P. furiosus ferredoxin for the monocations Cs+ and Tl+ in the absence of reducing agents. Both of these metal ions are large and polarizable, but they differ greatly in their propensity for ionic versus covalent interactions. The structural, electronic, and magnetic properties of the [Fe3S4](0,+) + cluster in P. furiosus ferredoxin in the presence of excess Cs+ and Tl+ were studied by EPR, magnetic circular dichroism, resonance Raman, and electron-nuclear double resonance spectroscopy. Magnetic circular dichroism and resonance Raman studies indicate that Tl+ but not Cs+ is incorporated into the reduced [Fe3S4](0) cluster with retention of the S = 2 (D < 0) ground state to yield a [TlFe3S(4)](+) cluster. EPR studies provide evidence for Tl+ incorporation into the oxidized S = 1/2 [Fe3S4](+) cluster as well. The native protein exhibits a broad EPR signal as a result of the distribution of g-values from multiple cluster conformations. In the presence of excess Tl+, a much narrower axial EPR signal is observed, indicating a single cluster conformation. Furthermore,Tl-203,Tl-205 hyperfine coupling was observed at both 9 and 35 GHz. The large coupling constant, A(Tl) approximate to 370 MHz (13 mT), indicates a covalent interaction associated with the formation of [TlFe3S4](2+). In contrast, the presence of excess Cs+ does not change the EPR spectrum, nor is Cs-133 hyperfine coupling observed, indicating a failure to incorporate this ion, However, Cs-133 electron-nuclear double resonance signals were observed with hyperfine and quadrupole couplings of A(Cs) approximate to 1.2 MHz, P-z approximate to 0.7 MHz. This, in conjunction with resonance Raman data, suggests that a Cs+ ion binds to a specific residue near the oxidized cluster. This is the first report of Cs-133 ENDOR in a biological system and suggests that this readily available nucleus could provide a valuable probe for Na+ or K+ binding in paramagnetic biomolecules.