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

The hyperthermophilic archaeon Pyrococcus furiosus contains a novel 4F e ferredoxin in which one Fe ion lacks cysteinyl coordination. This un ique Fe ion can be easily removed to yield protein containing a [Fe3S4 ](0) cluster. Under reducing conditions, this cluster can bind exogeno us 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, a nd magnetic properties of the [Fe3S4](0,+) + cluster in P. furiosus fe rredoxin in the presence of excess Cs+ and Tl+ were studied by EPR, ma gnetic circular dichroism, resonance Raman, and electron-nuclear doubl e resonance spectroscopy. Magnetic circular dichroism and resonance Ra man studies indicate that Tl+ but not Cs+ is incorporated into the red uced [Fe3S4](0) cluster with retention of the S = 2 (D < 0) ground sta te to yield a [TlFe3S(4)](+) cluster. EPR studies provide evidence for Tl+ incorporation into the oxidized S = 1/2 [Fe3S4](+) cluster as wel l. The native protein exhibits a broad EPR signal as a result of the d istribution of g-values from multiple cluster conformations. In the pr esence of excess Tl+, a much narrower axial EPR signal is observed, in dicating a single cluster conformation. Furthermore,Tl-203,Tl-205 hype rfine coupling was observed at both 9 and 35 GHz. The large coupling c onstant, A(Tl) approximate to 370 MHz (13 mT), indicates a covalent in teraction associated with the formation of [TlFe3S4](2+). In contrast, the presence of excess Cs+ does not change the EPR spectrum, nor is C s-133 hyperfine coupling observed, indicating a failure to incorporate this ion, However, Cs-133 electron-nuclear double resonance signals w ere observed with hyperfine and quadrupole couplings of A(Cs) approxim ate 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 resi due near the oxidized cluster. This is the first report of Cs-133 ENDO R in a biological system and suggests that this readily available nucl eus could provide a valuable probe for Na+ or K+ binding in paramagnet ic biomolecules.