Ferredoxin and ferredoxin–heme maquettes

Abstract

The immense complexity inherent in enzyme structure presents a daunting challenge to understanding how proteins fold and assemble cofactors to establish catalytic fitness. The problem of protein folding and secondary structure design has recently been approached with the stratagem of synthesizing scaled-down polypeptides (1–7). Relatively simple design rules have been established for the construction of tetra-α-helix bundles using either a minimalist design approach (3) or a binary patterning strategy (8). We are using designed tetra-α-helix bundles to address the intricacies of protein–cofactor interactions in polypeptides that incorporate biochemical cofactors (9–12). It is our goal to establish the rules for cofactor incorporation and function in these simplified systems to produce minimal, functional, synthetic proteins—i.e., molecular maquettes (9).

We designed and synthesized a tetra-helix bundle, H10H24 (Fig. ​(Fig.11B), that incorporates one to four hemes via bis-His ligation (9). This simplified heme–protein maquette provides, under physiological conditions, for the study of heme–protein interactions, the basis for the variety of function in natural heme proteins. The spectroelectrochemical properties of the hemes in H10H24 are characteristic of those found in b-type cytochromes, as designed, including redox cooperativity due to heme–heme interactions.

Figure 1

Modular design of the ferredoxin maquette. (A) molscript (13) model of the monomeric hexadecapeptide with bound [4Fe–4S]. (B) Molecular model of the prototype heme–protein maquette H10H24. (C) Computer representation of the modular design helix–loop–helix ferredoxin–heme maquette (HLH–FdM) with a [4Fe–4S] cluster and a pair of bound hemes. (D) Primary sequence alignment illustrating the modular peptide design approach. Given are the sequences for Peptococcus aerogenes Fd (from which the [4Fe–4S]-binding domain was extracted), the natural sequence ferredoxin maquette FdM, versions of the ferredoxin maquette with one, two, and three cysteines to alanine modifications, the glycine-modified ferredoxin maquette, and the HLH–FdM. All synthetic peptides were C-terminally amidated, and the HLH–FdM was N-terminally acetylated.

As widespread and biochemically diverse as the heme proteins, the ferredoxins and related iron sulfur proteins differ from heme proteins in that they display a multitude of cluster nuclearities and coordination geometries (14). The challenge evident in the synthesis of ferredoxin maquettes is to design a peptide that not only self-assembles iron and sulfur ions but also guides the formation of a single cluster architecture. The interaction of iron–sulfur clusters with thiolate ligands in organic solvents has been studied extensively, and, in fact, preassembled clusters have been incorporated into cysteine-containing peptides in dimethyl sulfoxide/water (80:20) (15). However, maquettes offer a tractable system in which to study the assembly of these biological cofactors in aqueous media to determine their delicate interplay with the surrounding heterogeneous chiral protein matrix, a major principle in the acquisition of biological specificity and activity. Additionally, the development of modular protein domains that can be spliced together to form multicofactor peptides offers a systematic approach to the design of more elaborate self-assembling biological structures.

Herein, we describe the design and synthesis of maquettes for ferredoxins containing multinuclear [4Fe–4S] clusters. Using a surprisingly short hexadecapeptide (16) [the creation of which was based on insight gained from inspection of natural proteins (17–22) and inorganic model complexes (23–29)], we have examined the incorporation of a tetra-nuclear iron–sulfur cluster into synthetic peptides under physiological conditions. By substituting the cysteine ligands with noncoordinating alanine residues and exchanging all of the intervening amino acids for glycine, while holding the cysteines in their consensus positions, we have investigated the contributions of each alteration to the assembly of the cluster.

Furthermore, strategies are presented to assemble both [4Fe–4S] and hemes in a tetra-α-helix bundle that demonstrate feasibility for the general synthesis of maquettes containing multiple, juxtaposed, redox cofactors such as hemes, iron–sulfur clusters, flavins, amino acid radicals, and quinones. These motifs are common to the catalytic sites of complex native oxidoreductases—e.g., the active site [4Fe–4S] cluster and siroheme of sulfite reductase (30).

Acknowledgments

Footnotes