N-Isotope effects on the Raman spectra of Fe_2S_2 ferredoxin and Rieske ferredoxin: evidence for structural rigidity of metal sites

摘要

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 Fe_2S_2 ferredoxins have been distinguished previously by isotopic substitution 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 Fe_2S_2 ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform ~(15)-labeling of Anabaena Fe_2S_2f ferredoxin results in shifts of -1 cm~(-1) in the Fe-S~t stretching modes at 181, 340, and 357 cm~(-1). The ~(15)N dependence is ascribed to kinematic coupling of the Fe-S(Cys stretch with deformations of the cysteine bach bone, including the amide nitrogen. No ~(15N dependence occurs for the v(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-mono-oxygenase system of Pseudomonas mendocina. Upon selective ~(15)-labeling of the a-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 v(Fe-S~t) and δ(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with ~(15)N-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 Fe_2S_2 and Rieske ferredoxins presumably arises from the rigidity of the protein 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.