Ultrafast X-ray Absorption Spectroscopy Reveals Excited-State Dynamics of B12 Coenzymes Controlled by the Axial Base.

Polarized time-resolved X-ray absorption spectroscopy at the Co K-edge is used to probe the excited-state dynamics and photolysis of base-off methylcobalamin and the excited-state structure of base-off adenosylcobalamin. For both molecules, the final excited-state minimum shows evidence for an expansion of the cavity around the Co ion by ca. 0.04 to 0.05 Å. The 5-coordinate base-off cob(II)alamin that is formed following photodissociation has a structure similar to that of the 5-coordinate base-on cob(II)alamin, with a ring expansion of 0.03 to 0.04 Å and a contraction of the lower axial bond length relative to that in the 6-coordinate ground state. These data provide insights into the role of the lower axial ligand in modulating the reactivity of B12 coenzymes.

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.

The Photoactive Excited State of the B12-Based Photoreceptor CarH.

We have used transient absorption spectroscopy in the UV-visible and X-ray regions to characterize the excited state of CarH, a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light. With visible excitation, a nanosecond-lifetime photoactive excited state is formed with unit quantum yield. The time-resolved X-ray absorption near edge structure difference spectrum of this state demonstrates that the excited state of AdoCbl in CarH undergoes only modest structural expansion around the central cobalt, a behavior similar to that observed for methylcobalamin rather than for AdoCbl free in solution. We propose a new mechanism for CarH photoreactivity involving formation of a triplet excited state. This allows the sensor to operate with high quantum efficiency and without formation of potentially dangerous side products. By stabilizing the excited electronic state, CarH controls reactivity of AdoCbl and enables slow reactions that yield nonreactive products and bypass bond homolysis and reactive radical species formation.

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.

Ultrafast XANES Monitors Femtosecond Sequential Structural Evolution in Photoexcited Coenzyme B12.

Polarized X-ray absorption near-edge structure (XANES) at the Co K-edge and broadband UV-vis transient absorption are used to monitor the sequential evolution of the excited-state structure of coenzyme B12 (adenosylcobalamin) over the first picosecond following excitation. The initial state is characterized by sub-100 fs sequential changes around the central cobalt. These are polarized first in the y-direction orthogonal to the transition dipole and 50 fs later in the x-direction along the transition dipole. Expansion of the axial bonds follows on a ca. 200 fs time scale as the molecule moves out of the Franck-Condon active region of the potential energy surface. On the same 200 fs time scale there are electronic changes that result in the loss of stimulated emission and the appearance of a strong absorption at 340 nm. These measurements provide a cobalt-centered movie of the excited molecule as it evolves to the local excited-state minimum.

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 the Excited State of Methylcobalamin Using Polarized Time-Resolved X-ray Absorption Spectroscopy.

We use picosecond time-resolved polarized X-ray absorption near-edge structure (XANES) measurements to probe the structure of the long-lived photoexcited state of methylcobalamin (MeCbl) and the cob(II)alamin photoproduct formed following photoexcitation of adenosylcobalamin (AdoCbl, coenzyme B12). For MeCbl, we used 520 nm excitation and a time delay of 100 ps to avoid the formation of cob(II)alamin. We find only small spectral changes in the equatorial and axial directions, which we interpret as arising from small (<∼0.05 Å) changes in both the equatorial and axial distances. This confirms expectations based on prior UV-visible transient absorption measurements and theoretical simulations. We do not find evidence for the significant elongation of the Co-C bond reported by Subramanian [ J. Phys. Chem. Lett. 2018 , 9 , 1542 - 1546 ] following 400 nm excitation. For AdoCbl, we resolve the difference XANES contributions along three unique molecular axes by exciting with both 540 and 365 nm light, demonstrating that the spectral changes are predominantly polarized along the axial direction, consistent with the loss of axial ligation. These data suggest that the microsecond "recombination product" identified by Subramanian et al. is actually the cob(II)alamin photoproduct that is produced following bond homolysis of MeCbl with 400 nm excitation. Our results highlight the pronounced advantage of using polarization-selective transient X-ray absorption for isolating structural dynamics in systems undergoing atomic displacements that are strongly correlated to the exciting optical polarization.

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.

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.

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.

Fibroblasts from long-lived rodent species exclude cadmium.

Resistance to the lethal effects of cellular stressors, including the toxic heavy metal cadmium (Cd), is characteristic of fibroblast cell lines derived from long-lived bird and rodent species, as well as cell lines from several varieties of long-lived mutant mice. To explore the mechanism of resistance to Cd, we used inductively coupled plasma mass spectroscopy to measure the rate of Cd uptake into primary fibroblasts of 15 rodent species. These data indicate that fibroblasts from long-lived rodent species have slower rates of Cd uptake from the extracellular medium than those from short-lived species. In addition, fibroblasts from short-lived species export more zinc after exposure to extracellular Cd than cells from long-lived species. Lastly, fibroblasts from long-lived rodent species have lower baseline concentrations of two redox-active metals, iron and copper. Our results suggest that evolution of longevity among rodents required adjustment of cellular properties to alter metal homeostasis and to reduce the toxic effects of heavy metals that accumulate over the course of a longer life span.

De novo-designed metallopeptides with type 2 copper centers: modulation of reduction potentials and nitrite reductase activities.

Enzymatic reactions involving redox processes are highly sensitive to the local electrostatic environment. Despite considerable effort, the complex interactions among different influential factors in native proteins impede progress toward complete understanding of the structure-function relationship. Of particular interest is the type 2 copper center Cu(His)3, which may act as an electron transfer center in peptidylglycine α-hydroxylating monooxygenase (PHM) or a catalytic center in copper nitrite reductase (CuNiR). A de novo design strategy is used to probe the effect of modifying charged amino acid residues around, but not directly bound to, a Cu(His)3 center embedded in three-stranded coiled coils (TRI-H)3 [TRI-H = Ac-G WKALEEK LKALEEK LKALEEK HKALEEK G-NH2]. Specifically, the peptide TRI-EH (=TRI-HK22E) alters an important lysine to glutamate just above the copper binding center. With a series of TRI-EH peptides mutated below the metal center, we use a variety of spectroscopies (EPR, UV-vis, XAS) to show a direct impact on the protonation equilibria, copper binding affinities, reduction potentials, and nitrite reductase activities of these copper-peptide complexes. The potentials at a specific pH vary by 100 mV, and the nitrite reductase activities range over a factor of 4 in rates. We also observe that the affinities, potentials, and catalytic activities are strongly influenced by the pH conditions (pH 5.8-7.4). In general, Cu(II) affinities for the peptides are diminished at low pH values. The interplay among these factors can lead to a 200 mV shift in reduction potential across these peptides, which is determined by the pH-dependent affinities of copper in both oxidation states. This study illustrates the strength of de novo protein design in elucidating the influence of ionizable residues on a particular redox system, an important step toward understanding the factors that govern the properties of this metalloenzyme with a goal of eventually improving the catalytic activity.

Zinc stabilization of prefibrillar oligomers of human islet amyloid polypeptide.

The aggregation of human islet amyloid polypeptide (hIAPP) has been linked to beta-cell death in type II diabetes. Zinc present in secretory granules has been shown to affect this aggregation. A combination of EXAFS, NMR, and AFM experiments shows that the influence of zinc is most likely due to the stabilization of prefibrillar aggregates of hIAPP.

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.

Geometric and electrostatic study of the [4Fe-4S] cluster of adenosine-5'-phosphosulfate reductase from broken symmetry density functional calculations and extended X-ray absorption fine structure spectroscopy.

Adenosine-5'-phosphosulfate reductase (APSR) is an iron-sulfur protein that catalyzes the reduction of adenosine-5'-phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X(~80)-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron-sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S](2+) state in both native and substrate-bound APSR while EXAFS data recorded at ~0.1 Å resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On the other hand, DFT calculations provide an insight into the subtle differences between the geometry of the cluster in the native and APS-bound forms of APSR. A comparison between models with and without the tandem cysteine pair coordination of the cluster suggests a role for the unique coordination in facilitating a compact geometric structure and "fine-tuning" the electronic structure to prevent reduction of the cluster. Further, calculations using models in which residue Lys144 is mutated to Ala confirm the finding that Lys144 serves as a crucial link in the interactions involving the [4Fe-4S] cluster and APS.

The interaction of mitochondrial iron with manganese superoxide dismutase.

Superoxide dismutase 2 (SOD2) is one of the rare mitochondrial enzymes evolved to use manganese as a cofactor over the more abundant element iron. Although mitochondrial iron does not normally bind SOD2, iron will misincorporate into Saccharomyces cerevisiae Sod2p when cells are starved for manganese or when mitochondrial iron homeostasis is disrupted by mutations in yeast grx5, ssq1, and mtm1. We report here that such changes in mitochondrial manganese and iron similarly affect cofactor selection in a heterologously expressed Escherichia coli Mn-SOD, but not a highly homologous Fe-SOD. By x-ray absorption near edge structure and extended x-ray absorption fine structure analyses of isolated mitochondria, we find that misincorporation of iron into yeast Sod2p does not correlate with significant changes in the average oxidation state or coordination chemistry of bulk mitochondrial iron. Instead, small changes in mitochondrial iron are likely to promote iron-SOD2 interactions. Iron binds Sod2p in yeast mutants blocking late stages of iron-sulfur cluster biogenesis (grx5, ssq1, and atm1), but not in mutants defective in the upstream Isu proteins that serve as scaffolds for iron-sulfur biosynthesis. In fact, we observed a requirement for the Isu proteins in iron inactivation of yeast Sod2p. Sod2p activity was restored in mtm1 and grx5 mutants by depleting cells of Isu proteins or using a dominant negative Isu1p predicted to stabilize iron binding to Isu1p. In all cases where disruptions in iron homeostasis inactivated Sod2p, we observed an increase in mitochondrial Isu proteins. These studies indicate that the Isu proteins and the iron-sulfur pathway can donate iron to Sod2p.

Synthesis, characterization, and in vitro testing of superparamagnetic iron oxide nanoparticles targeted using folic Acid-conjugated dendrimers.

Organic-coated superparamagnetic iron oxide nanoparticles (OC-SPIONs) were synthesized and characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. OC-SPIONs were transferred from organic media into water using poly(amidoamine) dendrimers modified with 6-TAMRA fluorescent dye and folic acid molecules. The saturation magnetization of the resulting dendrimer-coated SPIONs (DC-SPIONs) was determined, using a superconducting quantum interference device, to be 60 emu/g Fe versus 90 emu/g Fe for bulk magnetite. Selective targeting of the DC-SPIONs to KB cancer cells in vitro was demonstrated and quantified using two distinct and complementary imaging modalities: UV-visible and X-ray fluorescence; confocal microscopy confirmed internalization. The results were consistent between the uptake distribution quantified by flow cytometry using 6-TAMRA UV-visible fluorescence intensity and the cellular iron content determined using X-ray fluorescence microscopy.

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites.

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)4G-NH2] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M-S distances of 2.63 and 2.54 A, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS2 bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)3(3-) --> M(II)(TRI LXC)3(-). While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)2(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)3(-), the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)2 (+) at pH < 5 followed by a two-proton dissociation step (pK(a2)) yielding M(II)(TRI LXC)3(-). Pb(II)(TRI LXC)(HTRI LXC)2(+) converts to Pb(II)(TRI LXC)3(-) at slightly lower pH values than the corresponding Cd(II)-peptide complexes. In addition, Pb(II) displays a lower pK (a) of binding to the "d"-substituted peptide, (TRI L12C, pK(a2) = 12.0) compared with the "a"-substituted peptide, (TRI L16C, pK (a2) = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K(bind) = 3.2 x 10(7) M(-1)) compared with that with TRI L16C (K(bind) = 1.2 x 10(7) M(-1)) at pH > 8.