N-Isotope effects on the Raman spectra of Fe₂S₂ ferredoxin and Rieske ferredoxin: evidence for structural rigidity of metal sites

Abstract.

The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe₂S₂ ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins. Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe₂S₂ ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform ¹⁵N-labeling of Anabaena Fe₂S₂ ferredoxin results in shifts of –1 cm^–1 in the Fe-S^t stretching modes at 282, 340, and 357 cm^–1. The ¹⁵N dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen. No ¹⁵N dependence occurs for the ν(Fe-S^b) modes at 395 and 426 cm^–1. Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-monooxygenase system of Pseudomonas mendocina. Upon selective ¹⁵N-labeling of the α-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm^–1 all undergo shifts of –1 to –2 cm^–1, thereby identifying them as combinations of ν(Fe-S^t) and δ(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with ¹⁵N-histidine (¹⁵N in either the α1,δ1 or δ1,ε2 positions). Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety. The extensive kinematic coupling of iron ligand vibrations observed in both the Fe₂S₂ and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins. In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer.