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.

Unexpected plasticity of the quaternary structure of iron-manganese superoxide dismutases.

Protein 3D structure can be remarkably robust to the accumulation of mutations during evolution. On the other hand, sometimes a single amino acid substitution can be sufficient to generate dramatic and completely unpredictable structural consequences. In an attempt to rationally alter the preferences for the metal ion at the active site of a member of the Iron/Manganese superoxide dismutase family, two examples of the latter phenomenon were identified. Site directed mutants of SOD from Trichoderma reesei were generated and studied crystallographically together with the wild type enzyme. Despite being chosen for their potential impact on the redox potential of the metal, two of the mutations (D150G and G73A) in fact resulted in significant alterations to the protein quaternary structure. The D150G mutant presented alternative inter-subunit contacts leading to a loss of symmetry of the wild type tetramer, whereas the G73A mutation transformed the tetramer into an octamer despite not participating directly in any of the inter-subunit interfaces. We conclude that there is considerable intrinsic plasticity in the Fe/MnSOD fold that can be unpredictably affected by single amino acid substitutions. In much the same way as phenotypic defects at the organism level can reveal much about normal function, so too can such mutations teach us much about the subtleties of protein structure.

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.

An Interprotein Co-S Coordination Complex in the B12-Trafficking Pathway.

The CblC and CblD chaperones are involved in early steps in the cobalamin trafficking pathway. Cobalamin derivatives entering the cytoplasm are converted by CblC to a common cob(II)alamin intermediate via glutathione-dependent alkyltransferase or reductive elimination activities. Cob(II)alamin is subsequently converted to one of two biologically active alkylcobalamins by downstream chaperones. The function of CblD has been elusive although it is known to form a complex with CblC under certain conditions. Here, we report that CblD provides a sulfur ligand to cob(II)alamin bound to CblC, forming an interprotein coordination complex that rapidly oxidizes to thiolato-cob(III)alamin. Cysteine scanning mutagenesis and EPR spectroscopy identified Cys-261 on CblD as the sulfur donor. The unusual interprotein Co-S bond was characterized by X-ray absorption spectroscopy and visualized in the crystal structure of the human CblD thiolato-cob(III)alamin complex. Our study provides insights into how cobalamin coordination chemistry could be utilized for cofactor translocation in the trafficking pathway.

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.

Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle.

Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet-visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

Non-heme High-Spin {FeNO}6-8 Complexes: One Ligand Platform Can Do It All.

Heme and non-heme iron-nitrosyl complexes are important intermediates in biology. While there are numerous examples of low-spin heme iron-nitrosyl complexes in different oxidation states, much less is known about high-spin (hs) non-heme iron-nitrosyls in oxidation states other than the formally ferrous NO adducts ({FeNO}7 in the Enemark-Feltham notation). In this study, we present a complete series of hs-{FeNO}6-8 complexes using the TMG3tren coligand. Redox transformations from the hs-{FeNO}7 complex [Fe(TMG3tren)(NO)]2+ to its {FeNO}6 and {FeNO}8 analogs do not alter the coordination environment of the iron center, allowing for detailed comparisons between these species. Here, we present new MCD, NRVS, XANES/EXAFS, and Mössbauer data, demonstrating that these redox transformations are metal based, which allows us to access hs-Fe(II)-NO-, Fe(III)-NO-, and Fe(IV)-NO- complexes. Vibrational data, analyzed by NCA, directly quantify changes in Fe-NO bonding along this series. Optical data allow for the identification of a "spectator" charge-transfer transition that, together with Mössbauer and XAS data, directly monitors the electronic changes of the Fe center. Using EXAFS, we are also able to provide structural data for all complexes. The magnetic properties of the complexes are further analyzed (from magnetic Mössbauer). The properties of our hs-{FeNO}6-8 complexes are then contrasted to corresponding, low-spin iron-nitrosyl complexes where redox transformations are generally NO centered. The hs-{FeNO}8 complex can further be protonated by weak acids, and the product of this reaction is characterized. Taken together, these results provide unprecedented insight into the properties of biologically relevant non-heme iron-nitrosyl complexes in three relevant oxidation states.

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.