Histidine Ligated Iron-Sulfur Peptides.

Iron-sulfur clusters are thought to be ancient cofactors that could have played a role in early protometabolic systems. Thus far, redox active, prebiotically plausible iron-sulfur clusters have always contained cysteine ligands to the cluster. However, extant iron-sulfur proteins can be found to exploit other modes of binding, including ligation by histidine residues, as seen with [2Fe-2S] Rieske and MitoNEET proteins. Here, we investigated the ability of cysteine- and histidine-containing peptides to coordinate a mononuclear Fe2+ center and a [2Fe-2S] cluster and compare their properties with purified iron-sulfur proteins. The iron-sulfur peptides were characterized by UV-vis, circular dichroism, and paramagnetic NMR spectroscopies and cyclic voltammetry. Small (≤6 amino acids) peptides can coordinate [2Fe-2S] clusters through a combination of cysteine and histidine residues with similar reduction potentials as their corresponding proteins. Such complexes may have been important for early cell-like systems.

Characterization of [2Fe-2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods.

Many iron-sulfur proteins involved in cluster trafficking form [2Fe-2S]-cluster-bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe-2S]-cluster-bridged proteins and their transient cluster-transfer intermediates. The use of this mechanistic and protein-characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA-like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster-transfer reactions between the holo-dimers and apo-ferredoxin (FDX2) are monitored, and intermediate [2Fe-2S] species, such as (FDX2:GLRX5:[2Fe-2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe-2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron-sulfur-cluster-bridged protein complexes and transient iron-sulfur-cluster transfer intermediates.

Analysis of Structure-Activity Relationships Based on the Hepatitis†C Virus SLIIb Internal Ribosomal Entry Sequence RNA-Targeting GGHYRFK⋠Cu Complex.

New therapeutics for targeting the hepatitis C virus (HCV) have been released in recent years. Although they are less prone to resistance, they are still administered in cocktails as a combination of drugs targeting various aspects of the viral life cycle. Herein, we aim to contribute to an arsenal of new HCV therapeutics by targeting the HCV internal ribosomal entry sequence (IRES) RNA through the development of catalytic metallodrugs that function to degrade rather than inhibit the target molecule. Based on a previously characterized HCV IRES stem-loop IIb RNA-targeting metallopeptide Cu-GGHYrFK (1⋅Cu), an all-l analogue (3⋅Cu) and a series of additional complexes with single alanine substitutions in the targeting domain were prepared and screened to determine the influence each amino acid side chain on RNA localization and recognition, and catalytic reactivity toward the RNA. Additional substitutions of the tyrosine position in complex 3⋅Cu were also investigated. Good agreement between calculated and measured binding affinities provided support for in silico modeling of the SLIIb RNA binding site and correlations with RNA cleavage sites. Examination of the cleavage products from reaction of the Cu complexes with SLIIb provided mechanistic insights, with the first observation of the 5'-geminal diol and 5'-phosphopropenal as products through the use of a Cu⋅ATCUN catalytic motif. Together, the data yielded insights into structure-function relationships that will guide future optimization efforts.

Catalytic Metallodrugs: Substrate-Selective Metal Catalysts as Therapeutics.

Metal complexes that catalyze inactivation and degradation of biomolecular targets can be developed into novel therapeutics (catalytic metallodrugs) against a variety of diseases. Despite recent advances in the field, a lack of substrate selectivity is a major hindrance to the development of catalytic metallodrugs for application in clinical practice. Improved targeting can minimize nonselective activity and the potential for side effects. Herein, we focus on recent developments toward novel metal catalysts that exhibit substrate selectivity against a variety of therapeutically relevant biomolecules. Design strategies for developing selective catalytic metallodrugs are also highlighted.

Toward the design of a catalytic metallodrug: selective cleavage of G-quadruplex telomeric DNA by an anticancer copper-acridine-ATCUN complex.

Telomeric DNA represents a novel target for the development of anticancer drugs. By application of a catalytic metallodrug strategy, a copper-acridine-ATCUN complex (CuGGHK-Acr) has been designed that targets G-quadruplex telomeric DNA. Both fluorescence solution assays and gel sequencing demonstrate the CuGGHK-Acr catalyst to selectively bind and cleave the G-quadruplex telomere sequence. The cleavage pathway has been mapped by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) experiments. CuGGHK-Acr promotes significant inhibition of cancer cell proliferation and shortening of telomere length. Both senescence and apoptosis are induced in the breast cancer cell line MCF7.

Tackling antimicrobial stewardship through synergy and antimicrobial peptides.

The unrestricted use of antibiotics has led to rapid development of antibiotic resistance (AR) and renewed calls to address this serious problem. This review summarizes the most common mechanisms of antibiotic action, and in turn antibiotic resistance, as well as pathways to mitigate the harm. Focus is then turned to emerging antibiotic strategies, including antimicrobial peptides (AMPs), with a discussion of their modes of action, biochemical features, and potential challenges for their use as antibiotics. The role of synergy in antimicrobials is also examined, with a focus on the synergy of AMPs and other emerging interactions with synergistic potential.

Phenotypic continuum of NFU1-related disorders.

Bi-allelic variants in Iron-Sulfur Cluster Scaffold (NFU1) have previously been associated with multiple mitochondrial dysfunctions syndrome 1 (MMDS1) characterized by early-onset rapidly fatal leukoencephalopathy. We report 19 affected individuals from 10 independent families with ultra-rare bi-allelic NFU1 missense variants associated with a spectrum of early-onset pure to complex hereditary spastic paraplegia (HSP) phenotype with a longer survival (16/19) on one end and neurodevelopmental delay with severe hypotonia (3/19) on the other. Reversible or irreversible neurological decompensation after a febrile illness was common in the cohort, and there were invariable white matter abnormalities on neuroimaging. The study suggests that MMDS1 and HSP could be the two ends of the NFU1-related phenotypic continuum.

Cluster exchange reactivity of [2Fe-2S]-bridged heterodimeric BOLA1-GLRX5.

Mitochondrial BOLA1 is known to form a [2Fe-2S] cluster-bridged heterodimeric complex with mitochondrial monothiol glutaredoxin GLRX5; however, the function of this heterodimeric complex is unclear. Some reports suggest redundant roles for BOLA1 and a related protein, BOLA3, with both involved in the maturation of [4Fe-4S] clusters in a subset of mitochondrial proteins. However, a later report on the structure of BOLA1-GLRX5 heterodimeric complex demonstrated a buried cluster environment and predicted a redox role instead of the cluster trafficking role suggested for the BOLA3-GLRX5 heterodimeric complex. Herein, we describe a detailed kinetic study of relative cluster exchange reactivity involving heterodimeric complex of BOLA1 with GLRX5. By the use of CD spectroscopy, it is demonstrated that [2Fe-2S]-bridged BOLA1-GLRX5 can be readily formed by cluster uptake from donors such as ISCU or [2Fe-2S](GS)4 complex, but not from ISCA1 or ISCA2. Rapid holo-formation following delivery from [2Fe-2S](GS)4 supports possible physiological relevance in the cellular labile iron pool. Holo [2Fe-2S] BOLA1-GLRX5 heterodimeric complex is incapable of donating cluster to apo protein acceptors, providing experimental support for a nontrafficking role. Finally, we report the formation and reactivity of the holo [2Fe-2S]-bridged BOLA1 homodimer (lacking a partner GLRX). While the holo-heterodimer is thermodynamically more stable, by contrast the holo BOLA1 homodimer does demonstrate facile cluster exchange reactivity.

G-quadruplex targeting chemical nucleases as a nonperturbative tool for analysis of cellular G-quadruplex DNA.

G-quadruplex structures are associated with various biological activities, while in vivo evidence is essential to confirm the formation of G-quadruplexes inside cells. Most conventional agents that recognize G-quadruplex, including antibodies and small-molecule G-quadruplex ligands, either stabilize the G-quadruplex or prevent G-quadruplex unfolding by helicase, thereby artificially increasing the G-quadruplex levels in cells. Unambiguous study of G-quadruplexes at natural cellular levels requires agents that do not enhance the stability of G-quadruplex. Herein, we report the first example of nonperturbative chemical nucleases that do not influence the stability of G-quadruplex telomeric DNA but can selectively cleave G-quadruplex DNA over duplex DNA. These chemical nucleases can be readily taken up by cells and promote selective cleavage of telomeric DNA with low levels of nonselective DNA cleavage of other regions of the genome. The cleavage of G-quadruplex telomeric DNA by nonperturbative chemical nucleases confirms the formation of G-quadruplex telomeric DNA in live cells.

Kinetic and structural characterization of human mortalin.

Human mortalin is an Hsp70 chaperone that has been implicated in cancer, Alzheimer's and Parkinson's disease, and involvement has been suggested in cellular iron-sulfur cluster biosynthesis. However, study of this important human chaperone has been hampered by a lack of active material sufficient for biochemical characterization. Herein, we report the successful purification and characterization of recombinant human mortalin in Escherichia coli. The recombinant protein was expressed in the form of inclusion bodies and purified by Ni-NTA affinity chromatography. The subsequently refolded protein was confirmed to be active by its ATPase activity, a characteristic blue-shift in the fluorescence emission maximum following the addition of ATP, and its ability to bind to a likely physiological substrate. Single turnover kinetic experiments of mortalin were performed and compared with another Hsp70 chaperone, Thermotogamaritima DnaK; with each exhibiting slow ATP turnover rates. Secondary structures for both chaperones were similar by circular dichroism criteria. This work describes an approach to functional expression of human mortalin that provides sufficient material for detailed structure-function studies of this important Hsp70 chaperone.

Metallotherapeutics: novel strategies in drug design.

A new paradigm for drug activity is presented, which includes both recognition and subsequent irreversible inactivation of therapeutic targets. Application to both RNA and protein biomolecules has been demonstrated. In contrast to RNA targets that are subject to strand scission chemistry mediated by ribose H-atom abstraction, proteins appear to be inactivated either through oxidative damage to amino acid side chains around the enzyme active site, or by backbone hydrolysis.

In Vitro Studies of Cellular Iron-Sulfur Cluster Biosynthesis, Trafficking, and Transport.

Iron-sulfur clusters are metal cofactors that comprise the largest class of metalloproteins and are utilized for a wide variety of functions ranging from electron transport to DNA repair. These clusters and their respective cluster-binding proteins are highly conserved and are produced in the mitochondria via an evolutionarily conserved process for export to the cytosol and delivery to other organelles, including the nucleus. Disruption of the biosynthetic pathway results in a number of disease conditions that reflect the essential requirements of cluster function and trafficking within the cell. In vivo studies are limited in their ability to examine the detailed molecular mechanisms of protein-protein interactions, since they often focus on the downstream effects of protein depletion or mutation. As such, in vitro analyses are essential for defining the roles of specific Fe-S proteins in trafficking events and supporting in vivo analyses of disease conditions arising from aberrant Fe-S assembly and trafficking. In this chapter, we describe a variety of methods for the analysis of structure-function relationships in holo Fe-S cluster proteins, as well as monitoring the kinetics and molecular mechanisms of Fe-S cluster transfer.

Cytosolic iron-sulfur cluster transfer-a proposed kinetic pathway for reconstitution of glutaredoxin 3.

Iron-sulfur (Fe-S) clusters are ubiquitously conserved and play essential cellular roles. The mechanism of Fe-S cluster biogenesis involves multiple proteins in a complex pathway. Cluster biosynthesis primarily occurs in the mitochondria, but key Fe-S proteins also exist in the cytosol. One such protein, glutaredoxin 3 (Grx3), is involved in iron regulation, sensing, and mediating [2Fe-2S] cluster delivery to cytosolic protein targets, but the cluster donor for cytosolic Grx3 has not been elucidated. Herein, we delineate the kinetic transfer of [2Fe-2S] clusters into Grx3 from potential cytosolic carrier/scaffold proteins, IscU and Nfu, to evaluate a possible model for Grx3 reconstitution in vivo.

Duplications of an iron-sulphur tripeptide leads to the formation of a protoferredoxin.

Based on UV-Vis, NMR, and EPR spectroscopies and DFT and molecular dynamics calculations, a model prebiotic [2Fe-2S] tripeptide was shown to accept and donate electrons. Duplications of the tripeptide sequence led to a protoferredoxin with increased stability. Duplications of primitive peptides may have contributed to the formation of contemporary ferredoxins.

Insight into the recognition, binding, and reactivity of catalytic metallodrugs targeting stem loop†IIb of hepatitis†C IRES RNA.

The complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb (SLIIb) of the hepatitis C virus (HCV) internal ribosomal entry site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein we describe additional studies focused on understanding the cleavage mechanism as well as the relationship of catalyst configuration to structural recognition and site-selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI-TOF mass spectrometry, melting temperature determinations, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all-D-amino acid form of this metallopeptide, complex 2-Cu, are reported and compared with those of complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD value of 76 ± 3 nM, and Michaelis-Menten parameters of kcat =0.14 ± 0.01 min(-1) and KM =7.9 ± 1.2 µM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to that of the 1-Cu parent peptide, with an IC50 value of 1.9 ± 0.4 µM and cytotoxicity exceeding 100 µM. RT-PCR experiments confirm a significant decrease in HCV RNA levels in replicon assays for up to nine days when treated with complex 1-Cu in three-day dosing increments. This study shows the influence that the α-carbon stereocenter has for this new class of compounds, while detailed mass spectrometry and computational analyses provide new insight into the mechanisms of recognition, binding, and reactivity.

Redox chemistry of the Schizosaccharomyces pombe ferredoxin electron-transfer domain and influence of Cys to Ser substitutions.

Schizosaccharomyces pombe (Sp) ferredoxin contains a C-terminal electron transfer protein ferredoxin domain (etp(Fd)) that is homologous to adrenodoxin. The ferredoxin has been characterized by spectroelectrochemical methods, and Mössbauer, UV-Vis and circular dichroism spectroscopies. The Mössbauer spectrum is consistent with a standard diferric [2Fe-2S](2+) cluster. While showing sequence homology to vertebrate ferredoxins, the E°' and the reduction thermodynamics for etp(Fd) (-0.392 V) are similar to plant-type ferredoxins. Relatively stable Cys to Ser derivatives were made for each of the four bound Cys residues and variations in the visible spectrum in the 380-450 nm range were observed that are characteristic of oxygen ligated clusters, including members of the [2Fe-2S] cluster IscU/ISU scaffold proteins. Circular dichroism spectra were similar and consistent with no significant structural change accompanying these mutations. All derivatives were active in an NADPH-Fd reductase cytochrome c assay. The binding affinity of Fd to the reductase was similar, however, V(max) reflecting rate limiting electron transfer was found to decrease ~13-fold. The data are consistent with relatively minor perturbations of both the electronic properties of the cluster following substitution of the Fe-bond S atom with O, and the electronic coupling of the cluster to the protein.

NMR assignments of a stable processing intermediate of human frataxin.

Frataxin, a nuclear encoded protein targeted to the mitochondrial matrix, has recently been implicated as an iron chaperone that delivers Fe(II) to the iron-sulfur assembly enzyme ISU. During transport across the mitochondrial membrane, the N-terminal mitochondrial targeting sequence of frataxin is cleaved in a two-step process to produce the "mature" protein found within the matrix; however, N-terminally extended forms of the protein have also been observed in vivo as a result of processing deficiencies. Structural characterization studies of the mature human frataxin ortholog suggest the protein's N-terminus is predominately unfolded, in contrast to what has been observed for the yeast ortholog. Here we report the NMR assignments of a stable intermediate in the processing of human frataxin. These studies were completed to provide structural insight into editing events that lead to mature protein formation. This report also provides structural details of frataxin editing anomalies produced in vivo during altered protein processing events.

Control of reduction thermodynamics in [2Fe-2S] ferredoxins Entropy-enthalpy compensation and the influence of surface mutations.

The reaction thermodynamics for the one-electron reduction of the [2Fe-2S] cluster of both human ferredoxin and various surface point mutants, in which each of the negatively charged residues Asp72, Glu73, Asp76, and Asp79 were converted to Ala, have been determined by variable temperature spectroelectrochemical measurements. The above are conserved residues that have been implicated in interactions between the vertebrate-type ferredoxins and their redox partners. In all cases, and similar to other 2Fe-ferredoxins, the reduction potentials are negative as a result of both an enthalpic and entropic stabilization of the oxidized state. Although all Hs Fd mutants, with the exception of Asp72Ala, show slightly higher E degrees ' values than that of wild type Hs Fd, according to expectations for a purely electrostatic model, they exhibit changes in the H degrees '(rc) values that are electrostatically counter-intuitive. The observation of enthalpy-entropy compensation within the protein series indicates that the mutation-induced changes in H degrees '(rc) and S degrees '(rc) are dominated by reduction-induced solvent reorganization effects. Protein-based entropic effects are likely to be responsible for the low E degrees ' value of D72A.

Control of cytochrome C redox potential: axial ligation and protein environment effects.

Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E degrees ') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the E degrees ' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature E degrees ' measurements. In this way, we have found that (i) the decrease in E degrees ' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetylmethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases E degrees ' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall E degrees ' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.

Electron transfer from HiPIP to the photooxidized tetraheme cytochrome subunit of Allochromatium vinosum reaction center: new insights from site-directed mutagenesis and computational studies.

The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy. At ambient redox potential Eh = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 +/- 0.2) 10(5) M(-1) s(-1) at low ionic strength. Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCl. The role of Phe48, which lies on the external surface of HiPIP close to the [Fe4S4] cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role. This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair.