Co(II) Substitution Enhances the Esterase Activity of a de Novo Designed Zn(II) Carbonic Anhydrase.

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH-)]N(TRIL2WL23H)3 n+ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH-)]N(TRIL2WL23H)3 + peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

Watching Excited State Dynamics with Optical and X-ray Probes: The Excited State Dynamics of Aquocobalamin and Hydroxocobalamin.

Femtosecond time-resolved X-ray absorption (XANES) at the Co K-edge, X-ray emission (XES) in the Co Kß and valence-to-core regions, and broadband UV-vis transient absorption are combined to probe the femtosecond to picosecond sequential atomic and electronic dynamics following photoexcitation of two vitamin B12 compounds, hydroxocobalamin and aquocobalamin. Polarized XANES difference spectra allow identification of sequential structural evolution involving first the equatorial and then the axial ligands, with the latter showing rapid coherent bond elongation to the outer turning point of the excited state potential followed by recoil to a relaxed excited state structure. Time-resolved XES, especially in the valence-to-core region, along with polarized optical transient absorption suggests that the recoil results in the formation of a metal-centered excited state with a lifetime of 2-5 ps. This combination of methods provides a uniquely powerful tool to probe the electronic and structural dynamics of photoactive transition-metal complexes and will be applicable to a wide variety of systems.

Cu(I) Binding to Designed Proteins Reveals a Putative Copper Binding Site of the Human Line1 Retrotransposon Protein ORF1p.

Long interspersed nuclear elements-1 (L1) are autonomous retrotransposons that encode two proteins in different open reading frames (ORF1 and ORF2). The ORF1p, which may be an RNA binding and chaperone protein, contains a three-stranded coiled coil (3SCC) domain that facilitates the formation of the biologically active homotrimer. This 3SCC domain is composed of seven amino acid (heptad) repeats as found in native and designed peptides and a stammer that modifies the helical structure. Cysteine residues occur at three hydrophobic positions (2 a and 1 d sites) within this domain. We recently showed that the cysteine layers in ORF1p and model de novo designed peptides bind the toxic metalloid lead(II) with high affinities, a feature that had not been previously recognized. However, there is little understanding of how essential metal ions might interact with this metal binding domain. We have, therefore, investigated the copper(I) binding properties of analogous de novo designed 3SCCs that contain cysteine layers within the hydrophobic core. The results from UV-visible and X-ray absorption spectroscopy show that these designed peptides bind Cu(I) with high affinity in a pH-dependent manner. At pH 9, monomeric trigonal planar Cu(I)S3 centers are formed with 1 equiv of metal, while dinuclear centers form with a second equivalent of metal. At physiologic pH conditions, the dinuclear center forms cooperatively. These data suggest that ORF1p is capable of binding two copper ions to its tris(cysteine) layers. This has major implications for ORF1p coiled coil domain stability and dynamics, ultimately potentially impacting the resulting biological activity.

Open Reading Frame 1 Protein of the Human Long Interspersed Nuclear Element 1 Retrotransposon Binds Multiple Equivalents of Lead.

The human long interspersed nuclear element 1 (LINE1) has been implicated in numerous diseases and has been suggested to play a significant role in genetic evolution. Open reading frame 1 protein (ORF1p) is one of the two proteins encoded in this self-replicating mobile genetic element, both of which are essential for retrotransposition. The structure of the three-stranded coiled-coil domain of ORF1p was recently solved and showed the presence of tris-cysteine layers in the interior of the coiled-coil that could function as metal binding sites. Here, we demonstrate that ORF1p binds Pb(II). We designed a model peptide, GRCSL16CL23C, to mimic two of the ORF1p Cys3 layers and crystallized the peptide both as the apo-form and in the presence of Pb(II). Structural comparison of the ORF1p with apo-(GRCSL16CL23C)3 shows very similar Cys3 layers, preorganized for Pb(II) binding. We propose that exposure to heavy metals, such as lead, could influence directly the structural parameters of ORF1p and thus impact the overall LINE1 retrotransposition frequency, directly relating heavy metal exposure to genetic modification.

Nitrite reductase activity within an antiparallel de novo scaffold.

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3 binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3 binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3 binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

Traversing the Red-Green-Blue Color Spectrum in Rationally Designed Cupredoxins.

Blue copper proteins have a constrained Cu(II) geometry that has proven difficult to recapitulate outside native cupredoxin folds. Previous work has successfully designed green copper proteins which could be tuned blue using exogenous ligands, but the question of how one can create a self-contained blue copper site within a de novo scaffold, especially one removed from a cupredoxin fold, remained. We have recently reported a red copper protein site within a three helical bundle scaffold which we later revisited and determined to be a nitrosocyanin mimic, with a CuHis2CysGlu binding site. We now report efforts to rationally design this construct toward either green or blue copper chromophores using mutation strategies that have proven successful in native cupredoxins. By rotating the metal binding site, we created a de novo green copper protein. This in turn was converted to a blue copper protein by removing an axial methionine. Following this rational sequence, we have successfully created red, green, and blue copper proteins within an alpha helical fold, enabling comparisons for the first time of their structure and function disconnected from the overall cupredoxin fold.

Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation.

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper-binding scaffolds: H3 (His3 ), H4 (His4 ), H2 DH (His3 Asp with two His and one Asp in the same plane) and H3 D (His3 Asp with three His in the same plane) by using the scaffold of the de novo protein GRα3 D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good CuII -bound structural mimics of Cu-only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.

Probing a Silent Metal: A Combined X-ray Absorption and Emission Spectroscopic Study of Biologically Relevant Zinc Complexes.

As the second most common transition metal in the human body, zinc is of great interest to research but has few viable routes for its direct structural study in biological systems. Herein, Zn valence-to-core X-ray emission spectroscopy (VtC XES) and Zn K-edge X-ray absorption spectroscopy (XAS) are presented as a means to understand the local structure of zinc in biological systems through the application of these methods to a series of biologically relevant molecular model complexes. Taken together, the Zn K-edge XAS and VtC XES provide a means to establish the ligand identity, local geometry, and metal-ligand bond lengths. Experimental results are supported by correlation with density-functional-theory-based calculations. Combining these theoretical and experimental approaches will enable future applications to protein systems in a predictive manner.

Making or Breaking Metal-Dependent Catalytic Activity: The Role of Stammers in Designed Three-Stranded Coiled Coils.

While many life-critical reactions would be infeasibly slow without metal cofactors, a detailed understanding of how protein structure can influence catalytic activity remains elusive. Using de novo designed three-stranded coiled coils (TRI and Grand peptides formed using a heptad repeat approach), we examine how the insertion of a three residue discontinuity, known as a stammer insert, directly adjacent to a (His)3 metal binding site alters catalytic activity. The stammer, which locally alters the twist of the helix, significantly increases copper-catalyzed nitrite reductase activity (CuNiR). In contrast, the well-established zinc-catalyzed carbonic anhydrase activity (p-nitrophenyl acetate, pNPA) is effectively ablated. This study illustrates how the perturbation of the protein sequence using non-coordinating and non-acid base residues in the helical core can perturb metalloenzyme activity through the simple expedient of modifying the helical pitch adjacent to the catalytic center.

Methylated Histidines Alter Tautomeric Preferences that Influence the Rates of Cu Nitrite Reductase Catalysis in Designed Peptides.

Copper proteins have the capacity to serve as both redox active catalysts and purely electron transfer centers. A longstanding question in this field is how the function of histidine ligated Cu centers are modulated by δ vs ε-nitrogen ligation of the imidazole. Evaluating the impact of these coordination modes on structure and function by comparative analysis of deposited crystal structures is confounded by factors such as differing protein folds and disparate secondary coordination spheres that make direct comparison of these isomers difficult. Here, we present a series of de novo designed proteins using the noncanonical amino acids 1-methyl-histidine and 3-methyl-histidine to create Cu nitrite reductases where δ- or ε-nitrogen ligation is enforced by the opposite nitrogen's methylation as a means of directly comparing these two ligation states in the same protein fold. We find that ε-nitrogen ligation allows for a better nitrite reduction catalyst, displaying 2 orders of magnitude higher activity than the δ-nitrogen ligated construct. Methylation of the δ nitrogen, combined with a secondary sphere mutation we have previously published, has produced a new record for efficiency within a homogeneous aqueous system, improving by 1 order of magnitude the previously published most efficient construct. Furthermore, we have measured Michaelis-Menten kinetics on these highly active constructs, revealing that the remaining barriers to matching the catalytic efficiency ( kcat/ KM) of native Cu nitrite reductase involve both substrate binding ( KM) and catalysis ( kcat).

M-BLANK: a program for the fitting of X-ray fluorescence spectra.

The X-ray fluorescence data from X-ray microprobe and nanoprobe measurements must be fitted to obtain reliable elemental maps. The most common approach in many fitting programs is to initially remove a per-pixel baseline. Using X-ray fluorescence data of yeast and glial cells, it is shown that per-pixel baselines can result in significant, systematic errors in quantitation and that significantly improved data can be obtained by calculating an average blank spectrum and subtracting this from each pixel.

Incorporation of second coordination sphere D-amino acids alters Cd(II) geometries in designed thiolate-rich proteins.

We use a de Novo protein design strategy to demonstrate that the second coordination sphere of a metal site plays a key role in controlling coordination geometries of Cd(II)-tris-thiolate complexes. Specifically, we show that alteration of chirality within the core hydrophobic packing region of a three-stranded coiled coil (3SCC) can control the coordination number of Cd(II) by limiting steric encumbrance to the metal center. Within a specific class of 3SCCs [Ac-G-(LKALEEK) n -G-NH2], where n = 4 is TRI and n = 5 is GRAND, one L-Leu may be substituted by L-Cys to generate a planar tris-thiolate array capable of metal binding. In the native peptide containing only the L-configuration of leucine, the three-Cys ligand site leads to a mixture of 3- and 4-coordinate Cd(II). When the L-Leu above (toward the N-terminus) the tris-Cys site is substituted with D-Leu, solely a 3-coordinate structure [Cd(II)S3] was obtained. When D-Leu is located below (toward the C-terminus), a mixture of two coordination geometries, presumably Cd(II)S3O and Cd(II)S3O2, is observed, while substitution with D-Leu both above and below the tris-Cys plane yields a higher percentage of 4-coordinate Cd(II)S3O species. Thus, the use of D-amino acids around a metal's coordination sphere provides a powerful tool for controlling the properties of future designed metalloproteins.

Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata.

The superfamily of metallothioneins (MTs) combines a diverse group of metalloproteins, sharing the characteristics of rather low molecular weight and high cysteine content. The latter provides MTs with the capability to coordinate thiophilic metal ions, in particular those with a d 10 electron configuration. The sub-family of plant MT3 proteins is only poorly characterized and there is a complete lack of three-dimensional structure information. Building upon our previous results on the Musa acuminata MT3 (musMT3) protein, the focus of the present work is to understand the metal cluster formation process, the role of the single histidine residue present in musMT3, and the metal ion binding affinity. We concentrate our efforts on the coordination of ZnII and CdII ions, using CoII as a spectroscopic probe for ZnII binding. The overall protein-fold is analysed with a combination of limited proteolytic digestion, mass spectrometry, and dynamic light scattering. Histidine coordination of metal ions is probed with extended X-ray absorption fine structure spectroscopy and CoII titration experiments. Initial experiments with isothermal titration calorimetry provide insights into the thermodynamics of metal ion binding.

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein.

Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key ß-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, α3DChC2. (1) While this design demonstrated that a ß-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide α3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRANDα3D (GRα3D) with Δ Gu = -11.4 kcal/mol compared to the original design's -5.1 kcal/mol. Diffraction quality crystals were grown of GRα3D (a first for an α3D peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GRα3DChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs' hyperfine coupling constant from 160 to 127 × 10-4 cm-1. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41-Cu interaction leads to the α3DChC2 construct's red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.

Ultrafast X-ray Absorption Near Edge Structure Reveals Ballistic Excited State Structural Dynamics.

Polarized ultrafast time-resolved X-ray absorption near edge structure (XANES) allows characterization of excited state dynamics following excitation. Excitation of vitamin B12, cyanocobalamin (CNCbl), in the αß-band at 550 nm and the γ-band at 365 nm was used to uniquely resolve axial and equatorial contributions to the excited state dynamics. The structural evolution of the excited molecule is best described by a coherent ballistic trajectory on the excited state potential energy surface. Prompt expansion of the Co cavity by ca. 0.03 Å is followed by significant elongation of the axial bonds (>0.25 Å) over the first 190 fs. Subsequent contraction of the Co cavity in both axial and equatorial directions results in the relaxed S1 excited state structure within 500 fs of excitation.

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity.

Protein design is a useful strategy to interrogate the protein structure-function relationship. We demonstrate using a highly modular 3-stranded coiled coil (TRI-peptide system) that a functional type 2 copper center exhibiting copper nitrite reductase (NiR) activity exhibits the highest homogeneous catalytic efficiency under aqueous conditions for the reduction of nitrite to NO and H2 O. Modification of the amino acids in the second coordination sphere of the copper center increases the nitrite reductase activity up to 75-fold compared to previously reported systems. We find also that steric bulk can be used to enforce a three-coordinate CuI in a site, which tends toward two-coordination with decreased steric bulk. This study demonstrates the importance of the second coordination sphere environment both for controlling metal-center ligation and enhancing the catalytic efficiency of metalloenzymes and their analogues.

Polarized XANES Monitors Femtosecond Structural Evolution of Photoexcited Vitamin B12.

Ultrafast, polarization-selective time-resolved X-ray absorption near-edge structure (XANES) was used to characterize the photochemistry of vitamin B12, cyanocobalamin (CNCbl), in solution. Cobalamins are important biological cofactors involved in methyl transfer, radical rearrangement, and light-activated gene regulation, while also holding promise as light-activated agents for spatiotemporal controlled delivery of therapeutics. We introduce polarized femtosecond XANES, combined with UV-visible spectroscopy, to reveal sequential structural evolution of CNCbl in the excited electronic state. Femtosecond polarized XANES provides the crucial structural dynamics link between computed potential energy surfaces and optical transient absorption spectroscopy. Polarization selectivity can be used to uniquely identify electronic contributions and structural changes, even in isotropic samples when well-defined electronic transitions are excited. Our XANES measurements reveal that the structural changes upon photoexcitation occur mainly in the axial direction, where elongation of the axial Co-CN bond and Co-NIm bond on a 110 fs time scale is followed by corrin ring relaxation on a 260 fs time scale. These observations expose features of the potential energy surfaces controlling cobalamin reactivity and deactivation.

Development of a single-cell X-ray fluorescence flow cytometer.

An X-ray fluorescence flow cytometer that can determine the total metal content of single cells has been developed. Capillary action or pressure was used to load cells into hydrophilic or hydrophobic capillaries, respectively. Once loaded, the cells were transported at a fixed vertical velocity past a focused X-ray beam. X-ray fluorescence was then used to determine the mass of metal in each cell. By making single-cell measurements, the population heterogeneity for metals in the µM to mM concentration range on fL sample volumes can be directly measured, a measurement that is difficult using most analytical methods. This approach has been used to determine the metal composition of 936 individual bovine red blood cells (bRBC), 31 individual 3T3 mouse fibroblasts (NIH3T3) and 18 Saccharomyces cerevisiae (yeast) cells with an average measurement frequency of ∼4 cells min(-1). These data show evidence for surprisingly broad metal distributions. Details of the device design, data analysis and opportunities for further sensitivity improvement are described.

De novo design and characterization of copper metallopeptides inspired by native cupredoxins.

Using de novo protein design, we incorporated a copper metal binding site within the three-helix bundle α3D (Walsh et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5486-5491) to assess whether a cupredoxin center within an α-helical domain could mimic the spectroscopic, structural, and redox features of native type-1 copper (CuT1) proteins. We aimed to determine whether a CuT1 center could be realized in a markedly different scaffold rather than the native ß-barrel fold and whether the characteristic short Cu-S bond (2.1-2.2 Å) and positive reduction potentials could be decoupled from the spectroscopic properties (ε600 nm = 5000 M(-1) cm(-1)) of such centers. We incorporated 2HisCys(Met) residues in three distinct α3D designs designated core (CR), chelate (CH), and chelate-core (ChC). XAS analysis revealed a coordination environment similar to reduced CuT1 proteins, producing Cu-S(Cys) bonds ranging from 2.16 to 2.23 Å and Cu-N(His) bond distances of 1.92-1.99 Å. However, Cu(II) binding to the CR and CH constructs resulted in tetragonal type-2 copper-like species, displaying an intense absorption band between 380 and 400 nm (>1500 M(-1) cm(-1)) and A|| values of (150-185) × 10(-4) cm(-4). The ChC construct, which possesses a metal-binding site deeper in its helical bundle, yielded a CuT1-like brown copper species, with two absorption bands at 401 (4429 M(-1) cm(-1)) and 499 (2020 M(-1) cm(-1)) nm and an A|| value ∼30 × 10(-4) cm(-4) greater than its native counterparts. Electrochemical studies demonstrated reduction potentials of +360 to +460 mV (vs NHE), which are within the observed range for azurin and plastocyanin. These observations showed that the designed metal binding sites lacked the necessary rigidity to enforce the appropriate structural constraints for a Cu(II) chromophore (EPR and UV-vis); however, the Cu(I) structural environment and the high positive potential of CuT1 centers were recapitulated within the α-helical bundle of α3D.

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils.

One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)(3)(+/2+), where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu(T2) center of copper nitrite reductase. In Cu(I/II)(TRIL23H)(3)(+/2+), Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)(3) active site is characterized in both oxidation states, revealing similarities to the Cu(T2) site in the natural enzyme. The species Cu(II)(TRIL23H)(3)(2+) in aqueous solution can be reduced to Cu(I)(TRIL23H)(3)(+) using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)(3)(2+) acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.