A de novo designed 2[4Fe-4S] ferredoxin mimic mediates electron transfer.

[Fe-S] clusters, nature's modular electron transfer units, are often arranged in chains that support long-range electron transfer. Despite considerable interest, the design of biomimetic artificial systems emulating multicluster-binding proteins, with the final goal of integrating them in man-made oxidoreductases, remains elusive. Here, we report a novel bis-[4Fe-4S] cluster binding protein, DSD-Fdm, in which the two clusters are positioned within a distance of 12 Å, compatible with the electronic coupling necessary for efficient electron transfer. The design exploits the structural repeat of coiled coils as well as the symmetry of the starting scaffold, a homodimeric helical protein (DSD). In total, eight hydrophobic residues in the core of DSD were replaced by eight cysteine residues that serve as ligands to the [4Fe-4S] clusters. Incorporation of two [4Fe-4S] clusters proceeds with high yield. The two [4Fe-4S] clusters are located in the hydrophobic core of the helical bundle as characterized by various biophysical techniques. The secondary structure of the apo and holo proteins is conserved; further, the incorporation of clusters results in stabilization of the protein with respect to chemical denaturation. Most importantly, this de novo designed protein can mimic the function of natural ferredoxins: we show here that reduced DSD-Fdm transfers electrons to cytochrome c, thus generating the reduced cyt c stoichiometrically.

Determination of nonligand amino acids critical to [4Fe-4S]2+/+ assembly in ferredoxin maquettes.

The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single [4Fe-4S]2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the. CIACGAC. consensus ferredoxin core motif observed in natural protein sequences. Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little [4Fe-4S]2+/+ cluster, 6 and 7%, respectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC. or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7. Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino acids. Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of [4Fe-4S] cluster binding relative to that of the glycine variant. The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence analysis, suggesting they are applicable to both the de novo design and structure-based redesign of natural proteins.

Synthetic mutants of Clostridium pasteurianum ferredoxin: open iron sites and testing carboxylate coordination.

The entire polypeptide chains for two new Clostridium pasteurianum ferredoxin (Fd) mutants were prepared with the following site-specific substitutions: Cys11Asp and Cys11 alpha-aminobutyric acid (Cys11 alpha-Aba), the latter being a non-naturally occurring amino acid. Standard t-Boc procedures were used for the synthesis and the peptides. The two apoproteins were reconstituted to the 2[4Fe-4S] holoprotein and their spectroscopic, redox and thermal properties were compared with those of native C.pasteurianum Fds. The fully reconstituted Cys11Asp and Cys11 alpha-Aba mutants were initially found to have both clusters intact, i.e. they were 2[4Fe-4S] ferredoxins. The unconventional ligands of Asp and alpha-Aba led to holo-Fds that were not very stable and easily released an iron to form the [3Fe-4S] cluster, presumably through oxidation. The Cys11 alpha-Aba mutant was somewhat more thermally stable than Cys11Asp. In contrast, while both mutants were less stable than the native protein upon exposure to oxygen, the Cys11 alpha-Aba mutant was less stable than Cys11Asp. The Cys11Gly mutant was also prepared, but all attempts, despite repeated and varied experimental conditions, at reconstitution to the Cys11Gly holo 2[4Fe-4S] Fd were unsuccessful, probably because a Gly-Gly sequence is known to break structure. This work, when compared with molecular biological site-specific mutagenesis, shows some of the advantages of chemical/in vitro reconstitution: certain mutants which cannot be detected as holoproteins by site-specific mutagenesis can be formed after all in vitro. Nonetheless, it seems apparent that altering any of the Cys coordination sites of the Fd clusters results in fundamentally more unstable ferredoxins.

Total synthesis of a miniferredoxin.

A miniferredoxin has been designed based on the Desulfovibrio gigas ferredoxin II structure and has been successfully synthesized. The 31 amino acid apoprotein was synthesized via standard Fmoc solid phase peptide synthesis and in vitro cluster insertion carried out. The UV-visible spectrum of the miniferredoxin (peak at 300 nm and a shoulder at 405 nm) shows the same features as that of the D. gigas ferredoxin. Cyclic voltammetry indicated a quasireversible electrode process with a midpoint potential of -370mV vs NHE, which demonstrates that the miniferredoxin is redox active. From these and EPR studies, we propose the incorporation of a Fe4S4 cluster.

A synthetic analogue for the active site of plant-type ferredoxin: two different coordination isomers by a four-cys-containing [20]-peptide.

The (Fe2S2)2+ complex of an artificial 20-peptide ligand, Ac-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Pro-Leu-Leu-T hr-Cys- Val-NH2, containing an invariant Cys-A-B-C-D-Cys-X-Y-Cys (A, B, C, D, X, Y = amino acid residues) fragment of plant-type ferredoxins was synthesized by a ligand exchange method with [Fe2S2(S-t-Bu)4]2-. 1H-nmr spectroscopic and electrochemical data of the complex indicate the presence of two coordination isomers. One of them having a Cys-X-Y-Cys bridging coordination to the two Fe(III) ions, has the (Fe2S2)2+ core environment similar to those of the denatured plant-type ferredoxins and exhibits a positive shifted redox potential at -0.64 V vs saturated colonel electrode (SCE) in N,N-dimethylformamide (DMF). Another isomer with the Cys-A-B-C-D-Cys bridging coordination shows a negative redox potential at -0.96 V vs SCE in DMF.

A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.

The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.

Semisynthetic peptides and proteins.

Semisynthesis provides a flexible approach for using chemical synthesis to produce large, biologically active polypeptides and analogues. Currently developing improvements in the basic methods used, including polypeptide fragmentation, peptide synthesis, and reconstitution of synthetic and native components, make this overall approach applicable to a variety of species. Sequence modification through semisynthesis thus provides a flexible route to explore the code of rules whereby primary structure directs higher order properties of folded conformation and biological function of large peptides and proteins. The fruits of this endeavor, an understanding of how these macromolecules work, and therein, a basis for design of new structures that ultimately may be produced directly or by recombinant DNA methods, have begun to emerge.

Amino acids and peptides. XLII. Synthesis of an octapeptide sequence (A20-A27) of rubredoxin.

A protected octapeptide corresponding to a section found in the first half of the protein chain of rubredoxin has been prepared by standard peptide methods. Alternative approaches of this fragment are discussed in some detail. This work completes the series of subunits necessary to synthesize the molecule.