On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe–4S] ferredoxin

Abstract

The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] electron transfer proteins has not yet found a functional basis. The two such Pro of Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directed mutagenesis. Any one of them, but not both simultaneously, can be substituted without impairing the proper folding of the protein. The reduction potentials of the ferredoxin variants fall in a narrow range of < 20 mV above the potential of the native protein. The biological activities with C. pasteurianum hydrogenase and pyruvate-ferredoxin oxidoreductase do not change significantly, except when Lys replaces Pro. In these cases, the data suggest that the two clusters of 2[4Fe-4S] ferredoxin may not always be equivalent in the interaction with the redox partners. Destabilization of the structure has been observed as the consequence of the Pro19 or Pro48 substitutions. Using 2-D NMR, this effect has been associated with perturbations of both the hydrogen bond network and one amino acid side chain around the [4Fe-4S] clusters. Thus, the conserved Pro found in the binding motif of [4Fe-4S] clusters in proteins strongly stabilizes the active site but does not play an essential role in the mechanism of electron transfer.

Identification of Residues of Rhodobacter capsulatus Ferredoxin I Important for Its Interaction with Nitrogenase

“…This latter study brings strong experimental support to the theoretical prediction that the reduction potential of a given redox protein is closely related to its molecular charge (Rees, 1985). In the case of ferredoxins however, such a correlation is not obvious (Shen et al, 1994;Quinkal et al, 1994, and this study) and it is largely unknown what structural factors are at the origin of the great variation in redox potential observed among ferredoxins, despite the availability of several high-resolution X-ray structures (Backes et al, 1991). The microenvironment around the clusters, and especially the accessibility of solvent water to the clusters, has been suggested to be of major importance (Backes et al, 1991 ;Langen et al, 1992).…”

The Two [4Fe-4S] Clusters in Chromatium vinosumFerredoxin Have Largely Different Reduction Potentials

“…The plasmids were sequenced with the dideoxynucleotide termination method (14). The genes bearing the correct mutations were expressed in E. coli K38/ pGP1-2 (15), as described previously (4), and the synthesis of ferredoxin was checked by [ 35 S]cysteine labeling (16). For large scale (20 liters) production, cells were grown at 30°C until they reached an optical density measured at 600 nm of about 1.5.…”

“…At these high pH values, structural modifications of the protein must also be considered. The case of cluster II is interesting because, in contrast to cluster I, this group displays an increase of the redox potential with increasing pH opposite to the effect of electrostatic repulsion between the [Fe 4 S 4 (Cys) 4 ] 2Ϫ/3Ϫ cluster and nearby deprotonated residues, such as His-43.…”

Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form

“…However, although similarities with the D. ambi alens Fd exist, Da FdIII exhibits two peculiarities ; four Curie-temperature-dependent peaks and a larger number of hyperfine shifted peaks. These factors make the NMR characteristics of reconstituted ht Da FdIII unique, and it is possible that this results from the Da FdIII having a glutamate instead of a proline adjacent to CysIV in the binding motif of the transformable cluster [42,43].…”