Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by S(N)2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(II) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 A. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding 'lid' is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.

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A 16-amino acid residue peptide derived from a consensus motif of natural ferredoxins incorporates a tetranuclear iron sulfur cluster under physiological conditions. Successful assembly of the [4Fe-4S] 2؉/1؉ cluster within a monomeric peptide was demonstrated using size exclusion chromatography, UV-visible, visible CD, and cryogenic EPR spectroscopies. The robustness of [4Fe-4S] 2؉/1؉ formation was tested using peptides with either the ligating cysteine exchanged for alanine or with the intervening amino acids replaced by glycine. The small size of the peptide allows for modular incorporation into more complex protein structures. In one larger structure, we describe a tetra-␣-helix bundle that self-assembles both iron-sulfur clusters and hemes, thereby demonstrating feasibility for the general synthesis of maquettes containing multiple, juxtaposed redox cofactors. This is a motif common to the catalytic sites of native oxidoreductases.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-␣-helix bundles using either a minimalist design approach (3) or a binary patterning strategy (8). We are using designed tetra-␣-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. 1B), 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.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 cysteinecontaining peptides in dimethyl sulfoxide͞water (80:20) (15). However, maquettes offer a tractable system in which to study...

Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV. BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.