Coupling of zinc and GTP binding drives G-domain folding in Acinetobacter baumannii ZigA.

COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated "nutritional immunity." Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homolog, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii (Ab) ZigA coordinates ZnII using all three cysteines derived from the invariant CXCC motif to form an S3(N/O) coordination complex, a feature inconsistent with the ZnII-bound crystal structure of a distantly related COG0523 protein of unknown function from Escherichia coli, EcYjiA. The binding of ZnII and guanine nucleotides is thermodynamically linked in AbZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain tertiary structure as revealed by global thermodynamics measurements and hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS also reveals that the HDX behavior of the G2 (switch 1) loop is highly sensitive to nucleotide status and becomes more exchange labile in the GDP (product)-bound state. Significant long-range perturbation of local stability in both the G-domain and the C-terminal domain define a candidate binding pocket for a client protein that appears sensitive to nucleotide status (GDP versus GTP). We place these new insights into the structure, dynamics, and energetics of intermolecular metal transfer into the context of a model for AbZigA metallochaperone function.

Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii.

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M-1 with a functional "division of labor" among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10-14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.

Drosophila melanogaster frataxin: protein crystal and predicted solution structure with identification of the iron-binding regions.

Friedreich's ataxia (FRDA) is a hereditary cardiodegenerative and neurodegenerative disease that affects 1 in 50 000 Americans. FRDA arises from either a cellular inability to produce sufficient quantities or the production of a nonfunctional form of the protein frataxin, a key molecule associated with mitochondrial iron-sulfur cluster biosynthesis. Within the mitochondrial iron-sulfur cluster (ISC) assembly pathway, frataxin serves as an allosteric regulator for cysteine desulfurase, the enzyme that provides sulfur for [2Fe-2S] cluster assembly. Frataxin is a known iron-binding protein and is also linked to the delivery of ferrous ions to the scaffold protein, the ISC molecule responsible for the direct assembly of [2Fe-2S] clusters. The goal of this report is to provide structural details of the Drosophila melanogaster frataxin ortholog (Dfh), using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, in order to provide the foundational insight needed to understand the structure-function correlation of the protein. Additionally, NMR iron(II) titrations were used to provide metal contacts on the protein to better understand how it binds iron and aids its delivery to the ISC scaffold protein. Here, the structural and functional similarities of Dfh to its orthologs are also outlined. Structural data show that bacterial, yeast, human and Drosophila frataxins are structurally similar, apart from a structured C-terminus in Dfh that is likely to aid in protein stability. The iron-binding location on helix 1 and strand 1 of Dfh is also conserved across orthologs.

Pb(II) coordination to the nonclassical zinc finger tristetraprolin: retained function with an altered fold.

Tristetraprolin (TTP) is a nonclassical CCCH zinc finger (ZF) that plays a crucial role in regulating inflammation. TTP regulates cytokine mRNAs by specific binding of its two conserved ZF domains (CysX8CysX5CysX3His) to adenylate-uridylate-rich sequences (AREs) at the 3'-untranslated region, leading to degradation of the RNA. Dysregulation of TTP in animal models has demonstrated several cytokine-related syndromes, including chronic inflammation and autoimmune disorders. Exposure to Pb(II), a prevalent environmental toxin, is known to contribute to similar pathologies, in part by disruption of and/or competition with cysteine-rich metalloproteins. TTP's role during stress as a ubiquitous translational regulator of cell signaling (and dysfunction), which may underpin various phenotypes of Pb(II) toxicity, highlights the importance of understanding the interaction between TTP and Pb(II). The impact of Pb(II) binding on TTP's fold and RNA-binding function was analyzed via UV-Vis spectroscopy, circular dichroism, X-ray absorption spectroscopy, nuclear magnetic resonance spectroscopy, and fluorescence anisotropy. A construct containing the two ZF domains of TTP (TTP-2D) bound to Pb(II) with nanomolar affinity and exhibited a different geometry and fold in comparison to Zn2-TTP-2D. Despite the altered secondary structure, Pb(II)-substituted TTP-2D bound a canonical ARE sequence more selectively than Zn2-TTP-2D. Taken together, these data suggest that Pb(II) may interfere with proper TTP regulation and hinder the cell's ability to respond to inflammation.

Photocatalytic Hydrogen Evolution by a De Novo Designed Metalloprotein that Undergoes Ni-Mediated Oligomerization Shift.

De novo metalloprotein design involves the construction of proteins guided by specific repeat patterns of polar and apolar residues, which, upon self-assembly, provide a suitable environment to bind metals and produce artificial metalloenzymes. While a wide range of functionalities have been realized in de novo designed metalloproteins, the functional repertoire of such constructs towards alternative energy-relevant catalysis is currently limited. Here we show the application of de novo approach to design a functional H2 evolving protein. The design involved the assembly of an amphiphilic peptide featuring cysteines at tandem a/d sites of each helix. Intriguingly, upon NiII addition, the oligomers shift from a major trimeric assembly to a mix of dimers and trimers. The metalloprotein produced H2 photocatalytically with a bell-shape pH dependence, having a maximum activity at pH 5.5. Transient absorption spectroscopy is used to determine the timescales of electron transfer as a function of pH. Selective outer sphere mutations are made to probe how the local environment tunes activity. A preferential enhancement of activity is observed via steric modulation above the NiII site, towards the N-termini, compared to below the NiII site towards the C-termini.

Characteristics of the Isu1 C-terminus in relation to [2Fe-2S] cluster assembly and ISCU Myopathy.

Mitochondrial [2Fe-2S] cluster biosynthesis is driven by the coordinated activities of the Iron-Sulfur Cluster (ISC) pathway protein machinery. Within the ISC machinery, the protein that provides a structural scaffold on which [2Fe-2S] clusters are assembled is the ISCU protein in humans; this protein is referred to as the "Scaffold" protein. Truncation of the C-terminal portion of ISCU causes the fatal disease "ISCU Myopathy", which exhibits phenotypes of reduced Fe-S cluster assembly in cells. In this report, the yeast ISCU ortholog "Isu1" has been characterized to gain a better understanding of the role of the scaffold protein in relation to [2Fe-2S] assembly and ISCU Myopathy. Here we explored the biophysical characteristics of the C-terminal region of Isu1, the segment of the protein that is truncated on the human ortholog during the disease ISCU Myopathy. We characterized the role of this region in relation to iron binding, protein stability, assembly of the ISC multiprotein complex required to accomplish Fe-S cluster assembly, and finally on overall cell viability. We determined the Isu1 C-terminus is essential for the completion of the Fe-S cluster assembly but serves a function independent of protein iron binding.

Molecular characteristics of proteins within the mitochondrial Fe-S cluster assembly complex.

Iron-Sulfur (Fe-S) clusters are essential for life, as they are widely utilized in nearly every biochemical pathway. When bound to proteins, Fe-S clusters assist in catalysis, signal recognition, and energy transfer events, as well as additional cellular pathways including cellular respiration and DNA repair and replication. In Eukaryotes, Fe-S clusters are produced through coordinated activity by mitochondrial Iron-Sulfur Cluster (ISC) assembly pathway proteins through direct assembly, or through the production of the activated sulfur substrate used by the Cytosolic Iron-Sulfur Cluster Assembly (CIA) pathway. In the mitochondria, Fe-S cluster assembly is accomplished through the coordinated activity of the ISC pathway protein complex composed of a cysteine desulfurase, a scaffold protein, the accessory ISD11 protein, the acyl carrier protein, frataxin, and a ferredoxin; downstream events that accomplish Fe-S cluster transfer and delivery are driven by additional chaperone/delivery proteins that interact with the ISC assembly complex. Deficiency in human production or activity of Fe-S cluster containing proteins is often detrimental to cell and organism viability. Here we summarize what is known about the structure and functional activities of the proteins involved in the early steps of assembling [2Fe-2S] clusters before they are transferred to proteins devoted to their delivery. Our goal is to provide a comprehensive overview of how the ISC assembly apparatus proteins interact to make the Fe-S cluster which can be delivered to proteins downstream to the assembly event.

Molecular Details of the Frataxin-Scaffold Interaction during Mitochondrial Fe-S Cluster Assembly.

Iron-sulfur clusters are essential to almost every life form and utilized for their unique structural and redox-targeted activities within cells during many cellular pathways. Although there are three different Fe-S cluster assembly pathways in prokaryotes (the NIF, SUF and ISC pathways) and two in eukaryotes (CIA and ISC pathways), the iron-sulfur cluster (ISC) pathway serves as the central mechanism for providing 2Fe-2S clusters, directly and indirectly, throughout the entire cell in eukaryotes. Proteins central to the eukaryotic ISC cluster assembly complex include the cysteine desulfurase, a cysteine desulfurase accessory protein, the acyl carrier protein, the scaffold protein and frataxin (in humans, NFS1, ISD11, ACP, ISCU and FXN, respectively). Recent molecular details of this complex (labeled NIAUF from the first letter from each ISC protein outlined earlier), which exists as a dimeric pentamer, have provided real structural insight into how these partner proteins arrange themselves around the cysteine desulfurase, the core dimer of the (NIAUF)2 complex. In this review, we focus on both frataxin and the scaffold within the human, fly and yeast model systems to provide a better understanding of the biophysical characteristics of each protein alone and within the FXN/ISCU complex as it exists within the larger NIAUF construct. These details support a complex dynamic interaction between the FXN and ISCU proteins when both are part of the NIAUF complex and this provides additional insight into the coordinated mechanism of Fe-S cluster assembly.

Evaluation of the Physicochemical Properties of the Iron Nanoparticle Drug Products: Brand and Generic Sodium Ferric Gluconate.

Complex iron nanoparticle-based drugs are one of the oldest and most frequently administered classes of nanomedicines. In the US, there are seven FDA-approved iron nanoparticle reference drug products, of which one also has an approved generic drug product (i.e., sodium ferric gluconate (SFG)). These products are indicated for the treatment of iron deficiency anemia and are administered intravenously. On the molecular level, iron nanomedicines are colloids composed of an iron oxide core with a carbohydrate coating. This formulation makes nanomedicines more complex than conventional small molecule drugs. As such, these products are often referred to as nonbiological complex drugs (e.g., by the nonbiological complex drugs (NBCD) working group) or complex drug products (e.g., by the FDA). Herein, we report a comprehensive study of the physiochemical properties of the iron nanoparticle product SFG. SFG is the single drug for which both an innovator (Ferrlecit) and generic product are available in the US, allowing for comparative studies to be performed. Measurements focused on the iron core of SFG included optical spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), X-ray powder diffraction (XRPD), 57Fe Mössbauer spectroscopy, and X-ray absorbance spectroscopy (XAS). The analysis revealed similar ferric-iron-oxide structures. Measurements focused on the carbohydrate shell comprised of the gluconate ligands included forced acid degradation, dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and gel permeation chromatography (GPC). Such analysis revealed differences in composition for the innovator versus the generic SFG. These studies have the potential to contribute to future quality assessment of iron complex products and will inform on a pharmacokinetic study of two therapeutically equivalent iron gluconate products.

Unraveling the RNA Binding Properties of the Iron-Sulfur Zinc Finger Protein CPSF30.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a "zinc finger" protein that plays a crucial role in the transition of pre-mRNA to RNA. CPSF30 contains five conserved CCCH domains and a CCHC "zinc knuckle" domain. CPSF30 activity is critical for pre-mRNA processing. A truncated form of the protein, in which only the CCCH domains are present, has been shown to specifically bind AU-rich pre-mRNA targets; however, the RNA binding and recognition properties of full-length CPSF30 are not known. Herein, we report the isolation and biochemical characterization of full-length CPSF30. We report that CPSF30 contains one 2Fe-2S cluster in addition to five zinc ions, as measured by inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, and X-ray absorption spectroscopy. Utilizing fluorescence anisotropy RNA binding assays, we show that full-length CPSF30 has high binding affinity for two types of pre-mRNA targets, AAUAAA and polyU, both of which are conserved sequence motifs present in the majority of pre-mRNAs. Binding to the AAUAAA motif requires that the five CCCH domains of CPSF30 be present, whereas binding to polyU sequences requires the entire, full-length CPSF30. These findings implicate the CCHC "zinc knuckle" present in the full-length protein as being critical for mediating polyU binding. We also report that truncated forms of the protein, containing either just two CCCH domains (ZF2 and ZF3) or the CCHC "zinc knuckle" domain, do not exhibit any RNA binding, indicating that CPSF30/RNA binding requires several ZF (and/or Fe-S cluster) domains working in concert to mediate RNA recognition.

Role of Gold in Inflammation and Tristetraprolin Activity.

The zinc finger protein tristetraprolin (TTP) regulates inflammation by downregulating cytokine mRNAs. Misregulation results in arthritis, sepsis and cancer, and there is an interest in modulating TTP activity with exogenous agents. Gold has anti-inflammatory properties and has recently been shown to modulate the signaling pathway that produces TTP, suggesting that TTP may be a target of gold. The reactivity of [AuIII (terpy)Cl]Cl2 with TTP was investigated by UV/Vis spectroscopy, spin-filter inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy and native electrospray ionization mass spectrometry. AuIII was found to replace zinc in the protein active site in the reduced AuI form, with the AuI ion coordinated to two cysteine residues in a linear geometry. The replacement of ZnII with AuI results in loss of both secondary structure and RNA binding function. In contrast, when ZnII TTP is bound to its RNA target, no replacement of ZnII with AuI is observed, even in the presence of excess AuIII terpy. This discovery of differential reactivity of gold with TTP versus TTP/RNA offers a potential strategy for selective targeting with gold complexes to control inflammation.

Refinement of protein Fe(II) binding characteristics utilizing a competition assay exploiting small molecule ferrous chelators.

Iron is the most prevalent metal in biology. Its chemical and redox versatility allows it to direct activity of many Fe binding proteins. While iron's biological applications are diverse, challenges inherent in having Fe(II) present at high abundance means cells must ensure delivery to the correct recipient, while also ensuring its chemistry is regulated. Having a detailed understanding of the biophysical characteristics of a protein's iron binding characteristics allows us to understand general cellular metal homeostasis events. Unfortunately, most spectroscopic techniques available to measure metal binding affinity require protein be in a homogeneous state. Homogeneity creates an artificial environment when measuring metal binding since within cells numerous additional metal binding biomolecules compete with the target. Here we investigate commercially available Fe(II) chelators with spectral markers coupled to metal binding and release. Our goal was to determine their utility as competitors while measuring aspects of metal binding by apoproteins during a metal binding competition assay. Adding chelators during apoprotein metal binding mimics heterogeneous metal binding environments present in vivo, and provides a more realistic metal binding affinity measurement. Ferrous chelators explored within this report include: Rhod-5N, Magfura-2, Fura-4F, Fura-2, and TPA (Tris-(2-byridyl-methyl)amine; each forms a 1:1 complex with Fe(II) and combined cover a binding range of 5 orders of magnitude (micromolar to nanomolar Kd). These chelators were used to calibrate binding affinities for yeast and fly frataxin (Yfh1 and Dfh, respectively), involved in mitochondrial FeS cluster bioassembly.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

Unique roles of iron and zinc binding to the yeast Fe-S cluster scaffold assembly protein "Isu1".

Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical attributes of the "Isu1" scaffold assembly protein. As a member of a highly homologous protein family, Isu1 has sequence conservation between orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs have been shown to exist in both "disordered" and "structured" conformations, a structural architecture that is directly related to conformations utilized during Fe-S cluster assembly. During assembly, the scaffold helps direct the delivery and utilization of Fe(ii) and persulfide substrates to produce [2Fe-2S] clusters, however Zn(ii) binding alters the activity of the scaffold while at the same time stabilizes the protein in its structured state. Additional studies confirm Zn binds to the scaffold's Cys rich active site, and has an impact on the protein's ability to make Fe-S clusters. Understanding the interplay between Fe(ii) and Zn(ii) binding to Isu1 in vitro may help clarify metal loading events that occur during Fe-S cluster assembly in vivo. Here we determine the metal : protein stoichiometry for Isu1 Zn and Fe binding to be 1 : 1 and 2 : 1, respectively. As expected, while Zn binding shifts the Isu1 to its structured state, folding is not influenced by Fe(ii) binding. X-ray absorption spectroscopy (XAS) confirms Zn(ii) binds to the scaffold's cysteine rich active site but Fe(ii) binds at a location distinct from the active site. XAS results show Isu1 binding initially of either Fe(ii) or Zn(ii) does not significantly perturb the metal site structure of alternate metal. XAS confirmed that four scaffold orthologs bind iron as high-spin Fe(ii) at a site composed of ca. 6 oxygen and nitrogen nearest neighbor ligands. Finally, in our report Zn binding dramatically reduces the Fe-S cluster assembly activity of Isu1 even in the presence of frataxin. Given the Fe-binding activity we report for Isu1 and its orthologs here, a possible mechanism involving Fe(ii) transport to the scaffold's active site during cluster assembly has been considered.

Backbone resonance assignments and secondary structure of the apo-Drosophila melanogaster frataxin homolog (Dfh).

Friedreich's ataxia, the most prevalent hereditary ataxia, is caused by a patient's inability to produce a viable form of the protein frataxin. Frataxin plays an essential role in cellular iron regulation and has been shown to participate in the assembly of iron-sulfur (Fe-S) clusters under a variety of roles, including modulating persulfide production and directing Fe(II) delivery to the assembly scaffold protein. While the activity and structure of multiple eukaryotic frataxin orthologs have been characterized, the fly ortholog has numerous advantages over other orthologs with regards to protein stability, its activity towards Fe-S cluster assembly and its stability for forming stable proteins partner assemblies. Given the obvious advantages for studying the Drosophila melanogaster frataxin homolog (Dfh) over its orthologs, we have undertaken a structural characterization of apo-Dfh as the first step towards solving the solution structure of the protein alone and in complex with protein partners within the Fe-S cluster assembly pathway.

Simultaneous nitrosylation and N-nitrosation of a Ni-thiolate model complex of Ni-containing SOD.

Nitric oxide (NO) is used as a substrate analogue/spectroscopic probe of metal sites that bind and activate oxygen and its derivatives. To assess the interaction of superoxide with the Ni center in Ni-containing superoxide dismutase (NiSOD), we studied the reaction of NO+ and NO with the model complex, Et4N[Ni(nmp)(SPh-o-NH2-p-CF3)] (1; nmp2- = dianion of N-(2-mercaptoethyl)picolinamide; -SPh-o-NH2-p-CF3 = 2-amino-4-(trifluoromethyl)benzenethiolate) and its oxidized analogue 1ox , respectively. The ultimate products of these reactions are the disulfide of -SPh-o-NH2-p-CF3 and the S,S-bridged tetrameric complex [Ni4(nmp)4], a result of S-based redox activity. However, introduction of NO to 1 affords the green dimeric {NiNO}10 complex (Et4N)2[{Ni(κ2-SPh-o-NNO-p-CF3)(NO)}2] (2) via NO-induced loss of nmp2- as the disulfide and N-nitrosation of the aromatic thiolate. Complex 2 was characterized by X-ray crystallography and several spectroscopies. These measurements are in-line with other tetrahedral complexes in the {NiNO}10 classification. In contrast to the established stability of this metal-nitrosyl class, the Ni-NO bond of 2 is labile and release of NO from this unit was quantified by trapping the NO with a CoII-porphyrin (70-80% yield). In the process, the Ni ends up coordinated by two o-nitrosaminobenzenethiolato ligands to result in the structurally characterized trans-(Et4N)2[Ni(SPh-o-NNO-p-CF3)2] (3), likely by a disproportionation mechanism. The isolation and characterization of 2 and 3 suggest that: (i) the strongly donating thiolates dominate the electronic structure of Ni-nitrosyls that result in less covalent Ni-NO bonds, and (ii) superoxide undergoes disproportionation via an outer-sphere mechanism in NiSOD as complexes in the {NiNO}9/8 state have yet to be isolated.

Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria.

Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles.

Nanothermometry Reveals Calcium-Induced Remodeling of Myosin.

Ions greatly influence protein structure-function and are critical to health and disease. A 10, 000-fold higher calcium in the sarcoplasmic reticulum (SR) of muscle suggests elevated calcium levels near active calcium channels at the SR membrane and the impact of localized high calcium on the structure-function of the motor protein myosin. In the current study, combined quantum dot (QD)-based nanothermometry and circular dichroism (CD) spectroscopy enabled detection of previously unknown enthalpy changes and associated structural remodeling of myosin, impacting its function following exposure to elevated calcium. Cadmium telluride QDs adhere to myosin, function as thermal sensors, and reveal that exposure of myosin to calcium is exothermic, resulting in lowering of enthalpy, a decrease in alpha helical content measured using CD spectroscopy, and the consequent increase in motor efficiency. Isolated muscle fibers subjected to elevated levels of calcium further demonstrate fiber lengthening and decreased motility of actin filaments on myosin-functionalized substrates. Our results, in addition to providing new insights into our understanding of muscle structure-function, establish a novel approach to understand the enthalpy of protein-ion interactions and the accompanying structural changes that may occur within the protein molecule.

From micelles to bicelles: Effect of the membrane on particulate methane monooxygenase activity.

Particulate methane monooxygenase (pMMO) is a copper-dependent integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus (Mcc.) capsulatus (Bath) and Methylomicrobium (Mm.) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure, as observed by electron paramagnetic resonance (EPR) spectroscopy. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from Mm. alcaliphilum 20Z reveals a mononuclear copper center in the PmoB subunit and indicates that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from Mm. alcaliphilum 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment and provide valuable insight into pMMO function.

Cu+-specific CopB transporter: Revising P1B-type ATPase classification.

The copper-transporting P1B-ATPases, which play a key role in cellular copper homeostasis, have been divided traditionally into two subfamilies, the P1B-1-ATPases or CopAs and the P1B-3-ATPases or CopBs. CopAs selectively export Cu+ whereas previous studies and bioinformatic analyses have suggested that CopBs are specific for Cu2+ export. Biochemical and spectroscopic characterization of Sphaerobacter thermophilus CopB (StCopB) show that, while it does bind Cu2+, the binding site is not the prototypical P1B-ATPase transmembrane site and does not involve sulfur coordination as proposed previously. Most important, StCopB exhibits metal-stimulated ATPase activity in response to Cu+, but not Cu2+, indicating that it is actually a Cu+ transporter. X-ray absorption spectroscopic studies indicate that Cu+ is coordinated by four sulfur ligands, likely derived from conserved cysteine and methionine residues. The histidine-rich N-terminal region of StCopB is required for maximal activity, but is inhibitory in the presence of divalent metal ions. Finally, reconsideration of the P1B-ATPase classification scheme suggests that the P1B-1- and P1B-3-ATPase subfamilies both comprise Cu+ transporters. These results are completely consistent with the known presence of only Cu+ within the reducing environment of the cytoplasm, which should eliminate the need for a Cu2+ P1B-ATPase.