Interaction of T1⁺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]⁰cluster. Under reducing conditions, this cluster can bind exogenous metal dications, M²⁺(e.g., Ni²⁺and Zn²⁺), to yield [MFe₃S₄]⁺clusters. In this work, we have investigated the affinity of the [Fe₃S₄]^0,+cluster in P. furiosus ferredoxin for the monocations Cs⁺and T1⁺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 T1⁺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]⁰cluster with retention of the S = 2 (D< 0) ground state to yield a [T1Fe₃S₄]⁺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 T1⁺, a much narrower axial EPR signal is observed, indicating a single cluster conformation. Furthermore, ^203,205T1 hyperfine coupling was observed at both 9 and 35 GHz. The large coupling constant, A^T1≈ 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 ¹³³Cs hyperfine coupling observed, indicating a failure to incorporate this ion. However, ¹³³Cs electron-nuclear double resonance signals were observed with hyperfine and quadrupole couplings of A^Cs≈ 1.2 MHz, P_z≈ 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 ¹³³Cs 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.