Abstract
We have used
57
Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from
Pyrococcus furiosus
(
Pf
D14C Fd). To assist the normal mode assignments, we recorded the NRVS of D14C ferredoxin samples with
36
S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains: (Ph
4
P)
2
[Fe
4
S
4
Cl
4
]. Several distinct regions of NRVS intensity are identified, ranging from `protein' and torsional modes below 100 cm
−1
, through bending and breathing modes near 150 cm
−1
, to strong bands from Fe-S stretching modes between 250 cm
−1
and ~400 cm
−1
. The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra.
The
57
Fe partial vibrational densities of states (PVDOS) for the oxidized samples were interpreted by normal mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low frequency modes that may be relevant to electron transfer with
Pf
Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe
4
S
4
]
2+/1+
redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.