Normal Mode Analysis of Pyrococcus furiosus Rubredoxin via Nuclear Resonance Vibrational Spectroscopy (NRVS) and Resonance Raman Spectroscopy

Abstract

We have used ⁵⁷Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(S_cys)₄ site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). The oxidized form has also been investigated by resonance Raman spectroscopy. In the oxidized Rd NRVS, strong asymmetric Fe−S stretching modes are observed between 355 and 375 cm^-1; upon reduction these modes shift to 300−320 cm^-1. This is the first observation of Fe−S stretching modes in a reduced Rd. The peak in S−Fe−S bend mode intensity is at ∼150 cm^-1 for the oxidized protein and only slightly lower in the reduced case. A third band occurs near 70 cm^-1 for both samples; this is assigned primarily as a collective motion of entire cysteine residues with respect to the central Fe. The ⁵⁷Fe partial vibrational density of states (PVDOS) were interpreted by normal mode analysis with optimization of Urey−Bradley force fields. The three main bands were qualitatively reproduced using a D_2d Fe(SC)₄ model. A C₁ Fe(SCC)₄ model based on crystallographic coordinates was then used to simulate the splitting of the asymmetric stretching band into at least 3 components. Finally, a model employing complete cysteines and 2 additional neighboring atoms was used to reproduce the detailed structure of the PVDOS in the Fe−S stretch region. These results confirm the delocalization of the dynamic properties of the redox-active Fe site. Depending on the molecular model employed, the force constant K_Fe-S for Fe−S stretching modes ranged from 1.24 to 1.32 mdyn/Å. K_Fe-S is clearly diminished in reduced Rd; values from ∼0.89 to 1.00 mdyn/Å were derived from different models. In contrast, in the final models the force constants for S−Fe−S bending motion, H_S-Fe-S, were 0.18 mdyn/Å for oxidized Rd and 0.15 mdyn/Å for reduced Rd. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe−S proteins.