Dynamics of Rhodobacter capsulatus [2Fe-2S] Ferredoxin VI and Aquifex aeolicus Ferredoxin 5 via Nuclear Resonance Vibrational Spectroscopy (NRVS) and Resonance Raman Spectroscopy † (original) (raw)

We have used 57 Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe 2 S 2 (Cys) 4 sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe-S stretching modes peaking around 335 cm -1 , and additional features observed as high as the B 2u mode at ∼421 cm -1 . Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B 3u mode at ∼288 cm -1 , attributed to an asymmetric combination of the two FeS 4 breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS 4 breathing. NRVS and RR analysis of isotope shifts with 36 S-substituted into bridging S 2ions in Rc FdVI allowed quantitation of S 2motion in different normal modes. We observed the symmetric Fe-Fe stretching mode at ∼190 cm -1 in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm -1 . The 57 Fe partial vibrational densities of states (PVDOS) were interpreted by normalmode analysis with optimization of Urey-Bradley force fields. Progressively more complex D 2h Fe 2 S 2 S′ 4 , C 2h Fe 2 S 2 (SCC) 4 , and C 1 Fe 2 S 2 (Cys) 4 models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe-S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated for analysis of data on the reduced FdVI. Finally, the degree of collectivity was used to quantitate the delocalization of the dynamic properties of the redox-active Fe site. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins. † This work was funded by NIH Grants GM-65440 (to S.P.C.), EB-001962 (to S.P.C.), and GM-45303 (to T.I.), and the DOE Office of Biological and Environmental Research (to S.P.C.). Use of the APS is supported by the DOE Office of Basic Energy Sciences, Office of Science. SPring-8 is funded by JASRI.