Miniaturized metalloproteins: Application to iron-sulfur proteins (original) (raw)
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JBIC Journal of Biological Inorganic Chemistry, 2010
The design, synthesis, and metal-binding properties of DF3, a new de novo designed di-iron protein model are described ("DF" represents due ferri, Italian for "two iron," "di-iron"). DF3 is the latest member of the DF family of synthetic proteins. They consist of helix-loop-helix hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to those of non-heme carboxylate-bridged di-iron proteins. Unlike previous DF proteins, DF3 is highly soluble in water (up to 3 mM) and forms stable complexes with several metal ions (Zn, Co, and Mn), with the desired secondary structure and the expected stoichiometry of two ions per protein. UV-vis studies of Co(II) and Fe(III) complexes confirm a metal-binding environment similar to previous di-Co(II)- and di-Fe(III)-DF proteins, including the presence of a mu-oxo-di-Fe(III) unit. Interestingly, UV-vis, EPR, and resonance Raman studies suggest the interaction of a tyrosine adjacent to the di-Fe(III) center. The design of DF3 was aimed at increasing the accessibility of small molecules to the active site of the four-helix bundle. Indeed, binding of azide to the di-Fe(III) site demonstrates a more accessible metal site compared with previous DFs. In fact, fitting of the binding curve to the Hill equation allows us to quantify a 150% accessibility enhancement, with respect to DF2. All these results represent a significant step towards the development of a functional synthetic DF metalloprotein.
Proceedings of the National Academy of Sciences of the United States of America, 2000
De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 Å rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 Å. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation.
Protein …, 1999
Desulforedoxin~Dx!, isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric~2 ϫ 36 amino acids! protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin~Rd! type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In 3ϩ , Ga 3ϩ , Cd 2ϩ , Hg 2ϩ , and Ni 2ϩ salts. The In 3ϩ and Ga 3ϩ derivatives are isomorphous with the iron native protein; whereas Cd 2ϩ , Hg 2ϩ , and Ni 2ϩ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.
Molecules, 2021
Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetrahedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assembly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination ...
The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface
Journal of the American Chemical Society
Achieving fast electron transfer between a material and protein is a long-standing challenge confronting applications in bioelectronics, bioelectrocatalysis, and optobioelectronics. Interestingly, naturally occurring extracellular electron transfer proteins bind to and reduce metal oxides fast enough to enable cell growth, and thus could offer insight into solving this coupling problem. While structures of several extracellular electron transfer proteins are known, an understanding of how these proteins bind to their metal oxide substrates has remained elusive because this abiotic−biotic interface is inaccessible to traditional structural methods. Here, we use advanced footprinting techniques to investigate binding between the Shewanella oneidensis MR-1 extracellular electron transfer protein MtrF and one of its substrates, α-Fe 2 O 3 nanoparticles, at the molecular level. We find that MtrF binds α-Fe 2 O 3 specifically, but not tightly. Nanoparticle binding does not induce significant conformational changes in MtrF, but instead protects specific residues on the face of MtrF likely to be involved in electron transfer. Surprisingly, these residues are separated in primary sequence, but cluster into a small 3D putative binding site. This binding site is located near a local pocket of positive charge that is complementary to the negatively charged α-Fe 2 O 3 surface, and mutational analysis indicates that electrostatic interactions in this 3D pocket modulate MtrF−nanoparticle binding. Strikingly, these results show that binding of MtrF to α-Fe 2 O 3 follows a strategy to connect proteins to materials that resembles the binding between donor−acceptor electron transfer proteins. Thus, by developing a new methodology to probe protein−nanoparticle binding at the molecular level, this work reveals one of nature's strategies for achieving fast, efficient electron transfer between proteins and materials.
Conformational dependence of electron transfer across de novo designed metalloproteins
Proceedings of the National Academy of Sciences, 1996
Flash photolysis and pulse radiolysis measurements demonstrate a conformational dependence of electron transfer rates across a 16-mer helical bundle (three-helix metalloprotein) modified with a capping CoIII(bipyridine)3 electron acceptor at the N terminus and a 1-ethyl-1'-ethyl-4,4'- bipyridinium donor at the C terminus. For the CoIII(peptide)3-1-ethyl-1'-ethyl-4,4'-bipyridinium maquettes, the observed transfer is a first order, intramolecular process, independent of peptide concentration or laser pulse energy. In the presence of 6 M urea, the random coil bundle (approximately 0% helicity) has an observed electron transfer rate constant of kobs = 900 +/- 100 s-1. In the presence of 25% trifluoroethanol (TFE), the helicity of the peptide is 80% and the kobs increases to 2000 +/- 200 s-1. Moreover, the increase in the rate constant in TFE is consistent with the observed decrease in donor-acceptor distance in this solvent. Such bifunctional systems provide a class of m...
Journal of the …, 2005
The de novo protein DF1 is a minimal model for diiron and dimanganese metalloproteins, such as soluble methane monooxygenase. DF1 is a homodimeric four-helix bundle whose dinuclear center is formed by two bridging Glu side chains, two chelating Glu side chains, and two monodentate His ligands. Here, we report the di-Mn(II) and di-Co(II) derivatives of variants of this protein. Together with previously solved structures, 23 crystallographically independent four-helix bundle structures of DF1 variants have been determined, which differ in the bound metal ions and size of the active site cavity. For the di-Mn(II) derivatives, as the size of the cavity increases, the number and polarity of exogenous ligands increases. This collection of structures was analyzed to determine the relationship between protein conformation and the geometry of the active site. The primary mode of backbone movement involves a coordinated tilting and sliding of the first helix in the helix-loop-helix motif. Sliding depends on crystal-packing forces, the steric bulk of a critical residue that determines the dimensions of the active site access cavity, and the intermetal distance. Additionally, a torsional motion of the bridging carboxylates modulates the intermetal distance. This analysis provides a critical evaluation of how conformation, flexibility, and active site accessibility affect the geometry and ligand-binding properties of a metal center. The geometric parameters defining the DF structures were compared to natural diiron proteins; DF proteins have a restricted active site cavity, which may have implications for substrate recognition and chemical stability.
Structures of protein–protein complexes involved in electron transfer
Nature, 2013
Electron transfer (ET) reactions are essential for life since they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism 1. Key to these roles is the formation of transient inter-protein ET complexes. The structural basis for the control of specificity between partner proteins is lacking since these weak transient complexes have remained largely intractable for crystallographic studies 2,3. Inter-protein ET processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N 2 via the gaseous intermediates nitric oxide (NO) and nitrous oxide (N 2 O) when oxygen concentrations are limiting. The one electron reduction of nitrite to NO, a precursor to N 2 O, is performed by either a heme-or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome 4,5. Here we report the structures of the newly characterized three-domain hemec-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01Å resolution and its M92A and P93A mutains. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system i.e. where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 6 , and mutagenesis studies provide direct evidence for the importance of a hydrogen bonded water at the interface in ET. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding. Conversion of nitrate to N 2 known as the denitrification process is performed by a number of distinct enzymes 5,6. The first committed step, NO 2 − + 2H + + e − → NO + H 2 O is catalyzed by NiR. The well-studied two-domain CuNiRs are trimers of 106 kDa with each monomer having two domains with a characteristic β-sandwich curpredoxin motif. Each monomer has a type-1 Cu (T1Cu) center with (Cys-Met-His 2) ligation and a type-2 Cu (T2Cu) site with (His 3-H 2 O) ligation at the interface between two monomers, both of which
Metallomics, 2011
The peptide was prepared by solid phase peptide synthesis using the Fmoc methodology (Fmoc = 9-fluorenylmethoxycarbonyl). Rink Amide AM resin (Novabiochem, 200-400mesh, loading: 0.68 mmol/g) was used as a solid support. The amino acid building blocks were applied in 4-fold excess over the capacity of the resin. The amino acid residues were coupled to each other (and to the resin) by applying HBTU (3.8 eq./building block), HOBt (4 eq./building block) and N,N-diisopropylethylamine (7.8 eq./building block) in NMP. The Fmoc-protecting groups were removed by using a solution of 20% piperidine in NMP. The usual coupling reaction time was 1 h. The attachment of each amino acid residues was monitored by Kaiser-test (E. Kaiser,