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.

Effects of short- and long-term exposure of silver nanoparticles and silver ions to Nitrosomonas europaea biofilms and planktonic cells.

The increasing use of silver nanoparticles (AgNPs) in consumer products, and their resulting influx into wastewater, may pose a threat to biological nutrient removal in wastewater treatment plants. Planktonic ammonia-oxidizing bacteria (AOB), which convert ammonia to nitrite in the first step of nitrification, are highly sensitive to AgNPs and their released silver ions (Ag+), but the sensitivity of AOB biofilms to AgNPs and Ag+ is less clear. This study demonstrated that biofilms of Nitrosomonas europaea, a model AOB, were more resistant to both short-term and long-term exposure to AgNP and Ag+ than planktonic cells. The increased resistance of N. europaea biofilms was attributed primarily to the increased biomass and slower growth rates present in the biofilm. Similar inhibition mechanisms were observed for AgNPs and Ag+ in both planktonic cells and biofilms with enzymatic inhibition observed at lower concentrations and cell lysis observed at higher concentrations. Long-term continuous exposure to AgNPs lowered the inhibitory concentration by 1-2 orders of magnitude below that required by short-term exposures. Although the total AgNP load was similar between the short and long-term exposure scenarios, the long-term exposure resulted in an order of magnitude more silver being associated in the biofilms and is the primary reason for the increased sensitivity observed. This suggests that short-term batch toxicity assays may greatly underestimate the sensitivity of biofilm treatment systems to long-term exposures of low concentrations of AgNPs and Ag+.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with ß-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate.

The diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) requires a single electron reduction to convert the oxidized, as-isolated enzyme to an active conformation. We employ protein film voltammetry to investigate the mechanism of hydrogen peroxide turnover by So CcP. When the enzyme is poised in the active state by incubation with sodium l-ascorbate, the graphite electrode specifically captures a highly active state that turns over peroxide in a high potential regime. This is the first example of an on-pathway catalytic intermediate observed for a bacterial diheme cytochrome c peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome c peroxidase from Nitrosomonas europaea (Ne), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of So CcP confirms that the rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species centered on the low-potential heme, consistent with the same mutation in Ne CcP. The pH dependence of catalysis for wild-type So CcP suggests that reduction shifts the pK(a)'s of at least two amino acids. Mutation of His81 in "loop 1", a surface exposed loop thought to shift conformation during the reductive activation process, eliminated one of the pH dependent features, confirming that the loop 1 shifts, changing the environment of His81 during the rate-limiting step. The observed catalytic intermediate has the same electron stoichiometry and similar pH dependence to that previously reported for Ne CcP, which is constitutively active and therefore hypothesized to follow a different catalytic mechanism. The prominent similarities between the rate-limiting steps of differing mechanistic classes of bCcPs suggest unexpected similarities in the intermediates formed.

Global metabolomic responses of Nitrosomonas europaea 19718 to cold stress and altered ammonia feeding patterns.

The model ammonia-oxidizing bacterium Nitrosomonas europaea represents one of the environmentally and biotechnologically significant microorganisms. Genome-based studies over the last decade have led to many intriguing discoveries about its cellular biochemistry and physiology. However, knowledge regarding the regulation of overall metabolic routes in response to various environmental stresses is limited due to a lack of comprehensive, time-resolved metabolomic analyses. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling was performed to characterize the temporal variations of N. europaea 19718 intercellular metabolites in response to varied temperature (23 and 10 °C) and ammonia feeding patterns (shock loading and continuous feeding of 20 mg N/L). Approximately 87 metabolites were successfully identified and mapped to the existing pathways of N. europaea 19718, allowing interpretation of the influence of temperature and feeding pattern on metabolite levels. In general, varied temperature had a more profound influence on the overall metabolism than varied feeding patterns. Total extracellular metabolite concentrations (relative to internal standards and normalized to biomass weight) were lower under cold stress and shock loading conditions compared with the control (continuous feeding at 23 °C). Cold stress caused the widespread downregulation of metabolites involved in central carbon metabolism, amino acid, and lipid synthesis (e.g., malonic acid, succinic acid, putrescine, and phosphonolpyruvate). Metabolites that showed differences under varied feeding patterns were mainly involved in nucleotide acid, amino acid, and lipid metabolism (e.g., adenine, uracil, and spermidine). This study highlighted the roles of central carbon and nitrogen metabolism in countering cold stress and altered ammonia availability. In addition, transcriptomic, proteomic, and metabolomic data from three studies on N. europaea were compared to achieve a holistic view of some important synergy and interconnectivity among different cellular components (RNA, protein, and metabolites) during ammonia starvation.

Methionine ligand lability of homologous monoheme cytochromes c.

Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

Dissecting iron uptake and homeostasis in Nitrosomonas europaea.

The chemolithoautotroph Nitrosomonas europaea oxidizes about 25 mol of NH(3) for each mole of CO(2) that is converted to biomass using an array of heme and nonheme Fe-containing proteins. Hence mechanisms of efficient iron (Fe) uptake and homeostasis are particularly important for this Betaproteobacterium. Among nitrifiers, N.europaea has been the most studied to date. Characteristics that make N.europaea a suitable model to study Fe uptake and homeostasis are as follows: (a) its sequenced genome, (b) its capability to grow relatively well in 0.2 µM Fe in the absence of heterologous siderophores, and (c) its amenability to mutagenesis. In this chapter, we describe the methodology we use in our laboratory to dissect Fe uptake and homeostasis in the ammonia oxidizer N. europaea.

Liquid chromatography-mass spectrometry-based proteomics of Nitrosomonas.

During the last century, the research on aerobic ammonium-oxidizing bacteria (AOB) lead to many exciting physiological and biochemical discoveries. Nevertheless the molecular biology of AOB is not well understood. The availability of the genome sequences of several Nitrosomonas species opened up new possiblities to use state of the art transcriptomic and proteomic tools to study AOB. With the currect technology, thousands of proteins can be analyzed in several hours of measurement and translated proteins can be detected at femtomole and attomole concentrations. Moreover, it is possible to use mass spectrometry-based proteomics approach to analyze the expression, subcellular localization, posttranslational modifications, and interactions of translated proteins. In this chapter, we describe our LC-MS/MS methodology and quality control strategy to study the protein complement of Nitrosomonas eutropha C91.

Quantitative proteomic analysis of the chemolithoautotrophic bacterium Nitrosomonas europaea: comparison of growing- and energy-starved cells.

Obligately aerobic ammonia-oxidizing bacteria (AOB) like Nitrosomonas europaea play a pivotal role in the global nitrogen cycle. Although starvation tolerance is a key environmental adaptation, little is known about this response in AOB. The goal of these studies was to compare the composition of the N. europaea proteome in growing- and energy-starved cells using ¹5N labeling and HPLC-ESI-MS/MS. More than 6500 peptides were sequenced with high confidence, and matched to 876 proteins (34% of the protein coding genes). Of these, 126 proteins had two or more peptide forms identified by 10 or more scans, and were used in quantitative analysis and 27 were found to be significantly different in abundance between growing and starved cells. Proteins showing greater abundance in growing cells are geared toward biosynthesis, particularly DNA replication. Energy-starved cells were shifted away from biosynthesis and toward survival functions that included: cell envelope modification, protein protection/degradation, detoxification, and implementation of alternative energy generation mechanisms. Most of these activities have not previously been reported as associated with energy-starvation stress in N. europaea. This study provides insights into the potential effects of fluctuating environmental conditions on the regulation of physiological networks in N. europaea.

Structure of the first representative of Pfam family PF09410 (DUF2006) reveals a structural signature of the calycin superfamily that suggests a role in lipid metabolism.

The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively.

Computational prediction and transcriptional analysis of sRNAs in Nitrosomonas europaea.

Bacterial small noncoding RNAs (sRNAs) have been discovered in many genetically well-studied microorganisms and have been shown to regulate critical cellular processes at the post-transcriptional level. In this study, we used comparative genomics and microarray data to analyze the genome of the ammonia-oxidizing bacterium Nitrosomonas europaea for the presence and expression of sRNAs. Fifteen genes encoding putative sRNAs (psRNAs) were identified. Most of these genes showed altered expression in a variety of experimental conditions. The transcripts of two psRNAs were further characterized by mapping their 5'- and 3'-ends and by real-time PCR. The results of these analyses suggested that one of them, psRNA11, is involved in iron homeostasis in N. europaea.

Iron nutrition and physiological responses to iron stress in Nitrosomonas europaea.

Nitrosomonas europaea, as an ammonia-oxidizing bacterium, has a high Fe requirement and has 90 genes dedicated to Fe acquisition. Under Fe-limiting conditions (0.2 microM Fe), N. europaea was able to assimilate up to 70% of the available Fe in the medium even though it is unable to produce siderophores. Addition of exogenous siderophores to Fe-limited medium increased growth (final cell mass). Fe-limited cells had lower heme and cellular Fe contents, reduced membrane layers, and lower NH3- and NH2OH-dependent O2 consumption activities than Fe-replete cells. Fe acquisition-related proteins, such as a number of TonB-dependent Fe-siderophore receptors for ferrichrome and enterobactin and diffusion protein OmpC, were expressed to higher levels under Fe limitation, providing biochemical evidence for adaptation of N. europaea to Fe-limited conditions.

Classification of proteins based on minimal modular repeats: lessons from nature in protein design.

Proteins containing internal repeats within their primary sequence have received increased attention recently, as the extent of their presence in various organisms is recognized more fully, and their role in evolution is more thoroughly studied. Presented here is a technique used to detect and classify proteins based on a modular evolutionary phenomenon that results in a series of small internal repeats. The parameters chosen are based on a minimum segment of seven residues that result in simple functional scaffolds. The genomes and corresponding proteomes of a variety of eubacteria and archaea have been analyzed using an algorithm that searches prokaryotic genomes for proteins containing small conserved repeats assembled in a modular fashion similar to a recently characterized protein from the organism Nitrosomonas europaea. This analysis has revealed additional proteins present in N. europaea with similar modular characteristics. A further survey of a variety of organisms demonstrates that this evolutionary pathway has been utilized in other organisms as well, to yield a broad assortment of small modular proteins. A thorough description of the sequential characteristics of these modular proteins follows, along with a selection and discussion of the various proteins uncovered through this expanded search and analysis. Several databases of the proteins uncovered from this work and the program used to perform the search are available.

Spectroscopic and density functional studies of the red copper site in nitrosocyanin: role of the protein in determining active site geometric and electronic structure.

The electronic structure of the red copper site in nitrosocyanin is defined relative to that of the well understood blue copper site of plastocyanin by using low-temperature absorption, circular dichroism, magnetic circular dichroism, resonance Raman, EPR and X-ray absorption spectroscopies, combined with DFT calculations. These studies indicate that the principal electronic structure change in the red copper site is the sigma rather than the pi donor interaction of the cysteine sulfur with the Cu 3d(x2-y2) redox active molecular orbital (RAMO). Further, MCD data show that there is an increase in ligand field strength due to an increase in coordination number, whereas resonance Raman spectra indicate a weaker Cu-S bond. The latter is supported by the S K-edge data, which demonstrate a less covalent thiolate interaction with the RAMO of nitrosocyanin at 20% relative to plastocyanin at 38%. EXAFS results give a longer Cu-S(Cys) bond distance in nitrosocyanin (2.28 A) compared to plastocyanin (2.08 A) and also show a large change in structure with reduction of the red copper site. The red copper site is the only presently known blue copper-related site with an exogenous water coordinated to the copper. Density functional calculations reproduce the experimental properties and are used to determine the specific protein structure contributions to exogenous ligand binding in red copper. The relative orientation of the CuNNS and the CuSC(beta) planes (determined by the protein sequence) is found to be key in generating an exchangeable coordination position at the red copper active site. The exogenous water ligation at the red copper active site greatly increases the reorganization energy (by approximately 1.0 eV) relative to that of the blue copper protein site, making the red site unfavorable for fast outer-sphere electron transfer, while providing an exchangeable coordination position for inner-sphere electron transfer.

Characterization of a small metal binding protein from Nitrosomonas europaea.

A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.

Physiologic and proteomic evidence for a role of nitric oxide in biofilm formation by Nitrosomonas europaea and other ammonia oxidizers.

NO, a free radical gas, is the signal for Nitrosomonas europaea cells to switch between different growth modes. At an NO concentration of more than 30 ppm, biofilm formation by N. europaea was induced. NO concentrations below 5 ppm led to a reversal of the biofilm formation, and the numbers of motile and planktonic (motile-planktonic) cells increased. In a proteomics approach, the proteins expressed by N. europaea were identified. Comparison studies of the protein patterns of motile-planktonic and attached (biofilm) cells revealed several clear differences. Eleven proteins were found to be up or down regulated. Concentrations of other compounds such as ammonium, nitrite, and oxygen as well as different temperatures and pH values had no significant effect on the growth mode of and the proteins expressed by N. europaea.