Crystal structure of the 2[4Fe‐4S] ferredoxin from Chromatium vinosum: Evolutionary and mechanistic inferences for [3/4Fe‐4S] ferredoxins

Abstract

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatiurn vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 8, resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH. -23 H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn a-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.Keywords: Chromatium vinosum; crystal structure; electron transfer; evolution; ferredoxin; hydrogen bond; iron-sulfur Iron-sulfur proteins are increasingly emerging as a complex and diverse class of proteins fulfilling a number of different functions (Cammack, 1992). Ferredoxins with [4Fe-4S] clusters appear to be ubiquitous electron transfer proteins in most anaerobic microorganisms. Two main classes can be distinguished: one includes highreduction-potential (50-450 mV versus the normal hydrogen electrode or NHE) ferredoxins, which contain only one cluster; the other is composed of a more diverse group of proteins with one or two clusters exhibiting low reduction potentials in the range from -650 to -250 mV. A wealth of structural and spectroscopic data indicate that these two sets of proteins differ by the redox transition of the [4Fe-4S] cluster (Carter et al

The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H‐NMR spectroscopy

“…The latter resonance is characterized by its NOE to a resonance at 4.24 ppm, presumably Asp 23 Ha. This assignment is supported by the fact that the structurally characterized cubane ferredoxins share a turn involving the fourth cysteine of the cluster binding sequence (C(IV)PXX) as a highly conserved structural element [16][17][18][19][20][21][22][23]. Owing to this turn, a strong NOE connectivity is expected between CysIV H(3 2 and the Ha of amino acid i+3 with respect to CysIV (Asp 23 in the present case).…”

“…The overall tertiary structure of B. schlegeliis ferredoxin maintains the pseudo 2-fold symmetry of all dicluster ferredoxins [19,21,22,24,25]. This is evident from the similarity of the ID spectra of the two proteins and the observation of the expected NOE between Hf$i of CysIII of cluster I and H(3i of CysIII of cluster II in both cases.…”

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

“…The first set of variants involves modifications of the protein environment around cluster II which is coordinated by Cys-18, Cys-37, Cys-40, and Cys-49. In the variant referred to as ⌬1 the loop (residues 41-48) between two of the cysteine ligands of cluster II (10) has been replaced by two residues (Ala-Gln), therefore converting the sequence to a more conventional binding motif (CXXCAQC . .…”

“…In the K74Ϫ variant, half (residues 74 -82) of the C-terminal ␣-helix has been removed. In ⌬1K74Ϫ the two modifications were combined, and in K74E the buried hydrophilic side chain of lysine (10) has been substituted by glutamic acid. In two more variants, Y44C and Y44S, residue Tyr-44 of loop ⌬1 has been replaced by a cysteine or a serine.…”

“…It was found that, in contrast to other 2[4Fe-4S] ferredoxins without these elements, the electronic communication between the two clusters was apparently impaired (7). The gene encoding C. vinosum ferredoxin (CvFd) 1 was later cloned and expressed in Escherichia coli (9), and the structure of the protein was determined (10). These studies provide the necessary background for exploring the structure-function relationships in CvFd by protein engineering, and this report presents our initial efforts toward this aim.…”