A comparison of ferredoxin (Fd) crystal structures from Peptococcus aerogenes, Bacillus thermoproteolyticus, and Azotobacter vinelandii shows that the polypeptide chain folding in the vicinity of each of the [Fe4S4(Cys)4]2- clusters is highly conserved with regard to the location of the four cysteine ligands, the cysteine dihedral angles, and the eight amide NH groups that hydrogen bond to sulfur atoms of the cluster. Resonance Raman spectra of Fds from Clostridium pasteurianum and Clostridium acidi-urici exhibit a set of seven peaks assignable to Fe-S(bridging) vibrations and three peaks assignable to Fe-S(Cys) vibrations, as expected from the D2d distortion of the clusters observed in the X-ray structures. Hydrogen bonding of the bridging and terminal sulfur ligands has been verified by shifts in Fe-S vibrational frequencies in D2O. The solvent accessibility of the Fe4S4 clusters due to protein breathing motion is evident from the similarity of the isotope shifts between native Fd and protein equilibrated with deuterium prior to cluster reconstitution. Since the cluster geometry and hydrogen bonds are highly conserved in ferredoxins, their approximately 400-mV range in redox potentials must be due to different amino acid side chains in the vicinity of the clusters and to varying degrees of exposure to solvent. The crystal structure of the high-potential iron protein (HiPIP) from Chromatium vinosum reveals that, despite the presence of an [Fe4S4(Cys)4]2- cluster, there is no evolutionary relationship to the Fds in amino acid sequence, polypeptide chain folding, location of cysteine ligands, cysteine dihedral angles, or the five hydrogen bonds from amide NH groups. Resonance Raman spectra of reduced HiPIPs from Chromatium, Rhodocyclus, Rhodopila, and Ectothiorhodospira show a set of six Fe-S(b) (bridging) vibrations that are remarkably similar to those of the ferredoxins. These results lend strong support to X-ray crystallographic findings of similar D2d-distorted iron-sulfur clusters in Fds and HiPIPs and argue against the suggestion (Czernuszewicz, R. S.; Macor, K. A.; Johnson, M. K.; Gewirth, A.; Spiro, T. G. J. Am. Chem. Soc. 1987, 109, 7178) that HiPIPs have a more symmetric cluster geometry than Fds. The increased number and vibrational frequency of Fe-S(Cys) modes in HiPIPs is ascribed to coupling of Fe-S(Cys) stretching modes with S-C-C deformations, which is facilitated by the presence of an approximately 180-degrees Fe-S-gamma-C-beta-C-alpha dihedral angle in HiPIP. Deuterium exchange in C. vinosum HiPIP results in frequency shifts in the Fe-S(Cys) modes similar to those seen for Fds, consistent with the X-ray finding of four H bonds to cysteine sulfurs. However, in contrast to Fd, exchange of these hydrogen-bonded amide NH groups requires partial unfolding of the protein, confirming that the cluster in native HiPIP is inaccessible to water. The preference for the [Fe4S4(Cys)4]1- oxidation level (i.e., lower net charge on the cluster) in HiPIPs is ascribed primarily to the hydrophobic environment of the cluster and the smaller number of hydrogen bonds relative to ferredoxins.
