Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Biosynthesis of metal and oxide nanoparticles using Lactobacilli from yoghurt and probiotic spore tablets

Green, low-cost, and reproducible Lactobacillus-mediated biosynthesis of metal and oxide nanoparticles are reported. Silver and titanium dioxide nanoparticles are synthesized using Lactobacillus sp. procured from yoghurt and probiotic tablets. The synthesis is performed akin to room temperature in the laboratory ambience. X-ray and transmission electron microscopy analyses are performed to ascertain the formation of metallic and oxide nanoparticles. Individual nanoparticles having the dimensions of 10–25 nm (n-Ag) and 10–70 nm (n-TiO₂) are found. The mechanism involved for the synthesis of metallic and oxide nanoparticles has also been discussed.     

Soil C and N dynamics within a precipitation gradient in Mediterranean eucalypt plantations

Soil C and N dynamics were evaluated in five eucalypt plantations within a precipitation gradient (500–2,000 mm) in Portugal. Soil physical and chemical properties, total and labile (particulate organic matter, hydrolyzable, hot water soluble and microbial) soil C and N pools, and C and N mineralization were measured to characterize the C and N dynamics and their controlling factors within this gradient. Contents of total and labile soil organic C and N were positively correlated with the mean annual precipitation. A similar relationship was observed for net N mineralization (anaerobic and long-term aerobic incubation), gross N mineralization (¹⁵N isotope dilution technique) and C mineralization. In contrast, rates of C and N mineralization (per unit of C and N) were higher in the driest sites due to their higher proportion of particulate organic matter C. Net and gross N mineralization were strongly correlated and showed similar controlling factors (mean annual precipitation, total and labile C and N and extractable P contents), suggesting that net N mineralization during long-term aerobic incubation reflects gross N transformations. Although, gross NO₃–N production and gross NO₃–N immobilization were observed in all sites, net nitrification in the drier sites was not observed in the first weeks of the study. Our results suggest that, under Mediterranean conditions, mean annual precipitation is the major factor determining the C and N dynamics in soils with Eucalyptus plantations.     

The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide—Localization of a putative binding site

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high‐affinity titanium dioxide (TiO₂)–binding protein (TiBP), purified from Rhodococcus ruber , has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO₂‐binding capabilities with TiBP. Intrigued by the unique TiO₂‐binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti‐based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein‐TiO₂ surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO₂, indicating that neither the enzyme undergoes major conformational changes nor the TiO₂ binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO₂ through similar regions located far from the active site and the dimerization sites. The putative TiO₂‐binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal‐binding sites in proteins.     

Formation of titanium(IV) transferrin by reaction of human serum apotransferrin with titanium complexes

The reaction of human serum apotransferrin with titanium(IV) citrate under physiological conditions results in the formation of a specific bis-titanium(IV) transferrin adduct (Ti₂Tf hereafter) with two titanium(IV) ions loaded at the iron binding sites. The same specific Ti₂Tf complex is formed by reacting apotransferrin with titanium(III) chloride and exposing the sample to air. The derivative thus obtained was characterized by spectroscopic techniques, including absorption, UV difference, circular dichroism and ¹³C NMR spectroscopies, and shown to be stable within the pH range 5.5–9.0. Surprisingly, the reaction of apoTf with titanium(IV) nitrilotriacetate (NTA) does not lead to formation of appreciable amounts of Ti₂Tf, even after long incubation times, although some weak interactions of Ti(IV)NTA with apoTf are spectroscopically detected. Implications of the present results for a role of transferrin in the uptake, transport and delivery of soluble titanium(IV) compounds under physiological conditions are discussed.     

Small differences in ombrotrophy control regional-scale variation in methane cycling among <Emphasis Type="Italic">Sphagnum</Emphasis>-dominated peatlands

Across northern Alberta, Canada, bogs experience periodic wildfire and, in the Fort McMurray region, are exposed to increasing atmospheric N deposition related to oil sands development. As the fire return interval shortens and/or growing season temperatures increase, the regional peatland CO₂–C sink across northern Alberta will likely decrease, but the magnitude of the decrease could be diminished if increasing atmospheric N deposition alters N cycling in a way that stimulates post-fire successional development in bogs. We quantified net ammonification, nitrification, and dissolved organic N (DON) production in surface peat along a post-fire chronosequence of five bogs where we also experimentally manipulated N deposition (no water controls plus 0, 10, and 20 kg N ha^?1 yr^?1 simulated deposition, as NH₄NO₃). Initial KCl-extractable NH₄⁺–N, NO₃^?–N and DON averaged 176?±?6, 54?±?0.2, and 3580?±?40 ng N cm^?3, respectively, with no consistent changes as a function of time since fire and no consistent effects of experimental N addition. Net ammonification, nitrification, and DON production averaged 3.8?±?0.3, 1.6?±?0.2, and 14.3?±?2.0 ng N cm^?3 d^?1, also with no consistent changes as a function of time since fire and no consistent effects of experimental N addition. Our hypothesis that N mineralization would be stimulated after fire because root death would create a pulse of labile soil organic C was not supported, most likely because ericaceous plant roots typically are not killed in boreal bog wildfires. The absence of any N mineralization response to experimental N addition is most likely a result of rapid immobilization of added NH₄⁺–N and NO₃^?–N in peat with a wide C:N ratio. In these boreal bogs, belowground N cycling is likely characterized by large DON pools that turn over relatively slowly and small DIN pools that turn over relatively rapidly. For Alberta bogs that have persisted at historically low N deposition values and begin to receive higher N deposition related to anthropogenic activities, peat N mineralization processes may be largely unaffected until the peat C:N ratio reaches a point that no longer favors immobilization of NH₄⁺–N and NO₃^?–N.     

The size exclusion characteristics of type I collagen: implications for the role of noncollagenous bone constituents in mineralization

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril therefore provides the aqueous compartment in which mineral grows. Although knowledge of the size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding the mechanism of bone mineralization, there have been as yet no studies on the size exclusion properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we developed a gel filtration-like procedure that uses columns containing collagen from tendon and bone. The elution volumes of test molecules show the volume within the packed column that is accessible to the test molecules, and therefore reveal the size exclusion characteristics of the collagen within the column. These experiments show that molecules smaller than a 6-kDa protein diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa protein are excluded from this water. These studies provide an insight into the mechanism of bone mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all water within the fibril and so can directly impact mineralization. Although molecules larger than a 40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting apatite growth everywhere but within the fibril.     

Abiotic factors regulating nitrogen transformations in the organic layer of acid forest soils: Moisture and pH

The relationships between nitrogen transformations and moisture and pH in coniferous forest litter were determined using laboratory incubation experiments. A linear relation between gravimetric moisture content and nitrification was found within the whole studied range of moisture conditions (10–290% ODW). Net nitrogen mineralization increased linearly with moisture content up to 140% ODW. At higher moisture contents, net mineralization was found to be independent of moisture. Relative nitrification was found to be a linear function of moisture content. The dependence of the CO₂ production rates on moisture in the coniferous litter decreased from low to high moisture availability. Due to a nearly linear relationship between gravimetric moisture content and log-(water potential) within the investigated moisture range, the same type of relationships were found with this latter parameter as well. The relationship between nitrogen transformations and pH was studied by means of the addition of different amounts of HCl and NaOH during short incubation experiments (1 week). Nitrification was found to be a negative linear function of the H-ion concentration within the range of 0.04 (pH 4.40) and 0.36 (pH 3.45) mmol H-ion L^?1. At a higher H-ion concentration and thus at a lower pH than 3.45, no nitrate was produced any more. No relationship between net mineralization and pH was found.     

Adsorption of a PEO–PPO–PEO triblock copolymer on metal oxide surfaces with a view to reducing protein adsorption and further biofouling

Abstract

Biomolecule adsorption is the first stage of biofouling. The aim of this work was to reduce the adsorption of proteins on stainless steel (SS) and titanium surfaces by modifying them with a poly(ethylene oxide) (PEO)–poly(propylene oxide) (PPO)–PEO triblock copolymer. Anchoring of the central PPO block of the copolymer is known to be favoured by hydrophobic interaction with the substratum. Therefore, the surfaces of metal oxides were first modified by self-assembly of octadecylphosphonic acid. PEO–PPO–PEO preadsorbed on the hydrophobized surfaces of titanium or SS was shown to prevent the adsorption of bovine serum albumin (BSA), fibrinogen and cytochrome C, as monitored by quartz crystal microbalance (QCM). Moreover, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry were used to characterize the surfaces of the SS and titanium after competitive adsorption of PEO–PPO–PEO and BSA. The results show that the adsorption of BSA is well prevented on hydrophobized surfaces, in contrast to the surfaces of native metal oxides.     

Strategies Toward Stable Nonaqueous Alkali Metal–O2 Batteries

Alkali metal–O₂ batteries, by coupling high‐capacity alkali metal anodes with gaseous oxygen, possess extremely high gravimetric energy density that is comparable to gasoline and are potential energy storage technologies beyond lithium–ion batteries. The development of alkali metal–O₂ batteries has achieved great progress in recent years, from materials to prototype devices and on fundamental mechanisms. The stability of alkali metal–O₂ batteries is still poor, however, leading to a huge gap between laboratory research and commercial applications. A series of parasitic reactions result in the instability, which occur during electrochemical discharging and charging. The ubiquitous active oxygen species attack both the organic electrolyte and the carbon cathode, triggering various parasitic reactions. Meanwhile, dendrite growth and volume expansion upon repeated plating/stripping and O₂ crossover severely limit the reversibility of alkali metal anodes. Here, an overview of the strategies against these issues is given to improve the stability of nonaqueous alkali metal–O₂ batteries, which is discussed from three aspects: air cathodes, alkali metal anodes, and aprotic electrolytes. Furthermore, perspectives for future research of stable alkali metal–O₂ batteries are outlined.     

Fast mineralization of land-born C in inland waters: first experimental evidences of aquatic priming effect

In the context of global change, eroded soil carbon fate and its impact on aquatic ecosystems CO₂ emissions are subject to intense debates. In particular, soil carbon mineralization could be enhanced by its interaction with autochthonous carbon, a process called priming effect, but experimental evidences of this process are scarce. We measured in a microcosm experiment simulating oligo-mesotrophic and eutrophic aquatic conditions how quickly soil organic matter (SOM) sampled in diverse ecosystems was mineralized as compared to mineralization within soil horizons. For both nutrient loads, ¹³C-glucose was added to half of the microcosms to simulate exudation of labile organic matter (LOM) by phytoplankton. Effects of LOM on soil mineralization were estimated using the difference in δ¹³C between the SOM and the glucose. After 45 days of incubation, the mean SOM mineralization was 63% greater in the aquatic context, the most important CO₂ fluxes arising during the first days of incubation. Nutrients had no significant effect on SOM mineralization and glucose addition increased by 12% the mean SOM mineralization, evidencing the occurrence of a priming effect.     

Effect of seasonal freeze–thaw cycle on net nitrogen mineralization of soil organic layer in the subalpine/alpine forests of western Sichuan,China

Nitrogen mineralization, a main way that soil organic nitrogen converts to mineral nitrogen, is one of the key processes in soil nitrogen cycle. The mineral nitrogen has an important role in plant growth in the growing season. It has been widely accepted that soil freezing in winter can kill a number of microorganisms, weakening soil nitrogen mineralization. However, more and more recent studies have documented that soil microorganisms still have high activity during the deep freezing period, and obvious nitrogen mineralization in winter. Seasonal freeze–thaw cycle is a common phenomenon in the subalpine/alpine forest region, which may have a strong effect on soil ecological processes. Furthermore, the changing pattern of seasonal freeze–thaw cycles might have a significant influence on soil nitrogen mineralization in this region in the scenarios of global warming. As yet, little attention has been given to nitrogen mineralization of soil organic layer as affected by changed seasonal freeze–thaw pattern, although the increasing studies have demonstrated that winter warming might give strong effects on the litter decomposition and microbial activity in the subalpine/alpine forest regions. Therefore, a method of intact soil core incubation in combination with natural environmental gradient was employed by transferring forest soils from 3582 m (A₁) of altitude to 3298 m (A₂) of altitude and 3023 m (A₃) of altitude in the subalpine/alpine forests of western Sichuan, respectively. The amounts and rates of net nitrogen mineralization in soil organic layer were measured. The incubation period included the growing season and the freeze–thaw season from May 24, 2010 to April 19, 2011. The results suggested that significant net nitrogen mineralization was only observed in soil organic layer at low altitude (A₃) during the whole incubation period. Forest soils at higher altitudes (A₁ and A₂) showed obvious soil nitrogen immobilization. In comparison with the growing season which showed remarkable nitrogen immobilization characteristic, the freeze–thaw season showed obvious nitrogen mineralization at lower altitudes (A₂ and A₃). In contrast, the nitrogen immobilization amounts at high altitude (A₁) in freeze–thaw period were less than those in the growing season. Besides, the maximum of net nitrogen mineralization amounts and rates at high altitude (A₁) in soil organic layer mainly occurred in the late stage of growing season and the onset of freezing, soil nitrogen mineralization at the middle altitude (A₂) mainly occurred in the onset of freezing and the deep freezing period, while the highest amount and rate of net nitrogen mineralization at low altitude (A₃) occurred in the early stage of thawing and the late stage of growing season. Furthermore, the amount and rate of soil net nitrogen mineralization during the freeze–thaw season were increasing with the decrease of altitude, which correlated with soil freeze–thaw cycle and freezing process at different altitudes. These results indicated that increasing soil temperature in the future could not only significantly enhance soil nitrogen mineralization in the freeze–thaw season, but also improve soil nitrogen mineralization by increasing freeze–thaw cycle times and shortening freeze–thaw period. However, the processes were significantly influenced by soil micro-environment of subalpine/alpine forest regions.     

Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.     

Metalloenzymes and signal transduction: the protein serine/threonine phosphatases,a novel class of binuclear metal-containing enzymes

The serine/threonine protein phosphatases are important regulatory enzymes involved in signal transduction pathways in eukaryotic organisms. These enzymes include protein phosphatases 1, 2A, and 2B (also known as calcineurin). Recent structural data have indicated that the serine/threonine protein phosphatases are novel metalloenzymes containing a dinuclear metal ion cofactor at the active site. The dinuclear metal site is situated in a unique protein fold, a β-α-β-α-β motif which provides the majority of ligands to the metal ions. A similar fold is also seen in plant purple acid phosphatases, which also contain a dinuclear iron–zinc cofactor. In these enzymes, the two metal ions are bridged by a solvent molecule and a carboxylate group from an aspartic acid residue, juxtaposing the two metal ions to within 3.0–4.0?Å of each other. A similar motif has been identified in a number of other enzymes which exhibit phosphoesterase activity, implicating several of them as metalloenzymes which contain dinuclear metal ion cofactors.     

A metal‐chelate affinity reverse micellar system for protein extraction

A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X‐45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X‐45 less than 0.6, thermodynamically stable reverse micelles of water content (W₀) up to 30 are formed. Di(2‐ethylhexyl) phosphoric acid (HDEHP; 1–2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine‐rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper‐chelate reverse micelles (Cu‐RM) are characterized for their W₀, hydrodynamic radius (R_h), and aggregation number (N_ag). Similar with reverse micelles of bis‐2‐ethylhexyl sodium sulfosuccinate (AOT), R_h of the Cu‐RM is also linearly related to W₀. However, N_ag is determined to be 30–90 at W₀ of 5–30, only quarter to half of the AOT reverse micelles. Then, selective metal‐chelate extraction of histidine‐rich protein (myoglobin) by the Cu‐RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine‐tagged proteins expressed as inclusion bodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Presence of ferric hydroxide clusters in mutants of Haemophilus influenzae ferric ion-binding protein A

The periplasmic iron binding protein plays an essential role in the iron uptake pathway of Gram-negative pathogenic bacteria from the Pasteurellaceae and Neisseriaceae families and is critical for survival of these pathogens within the host. In this study, we report the crystal structures of two mutant forms of ferric ion-binding protein A (FbpA) from Haemophilus influenzae with bound multinuclear oxo-metal clusters. Crystals of site-directed mutants in the metal or anion binding ligands contain protein in the open conformation, and two mutant FbpAs, H9A and N175L, contain different cluster arrangements in the iron-binding pocket. The iron clusters are anchored by binding to the two tyrosine ligands (Tyr195 and Tyr196) positioned at the vertex of the iron-binding pocket but are not coordinated by the other metal binding ligands. Our results suggest that the metal clusters may have formed in situ, suggesting that the mutant FbpAs may serve as a simple model for protein-mediated mineralization.     

Toward Promising Cathode Catalysts for Nonlithium Metal–Oxygen Batteries

The success of Li–air/O₂ batteries has brought extensive attention to the development of various promising non‐Li metal–O₂ batteries, such as Zn–O₂, Al–O₂, Mg–O₂ batteries, etc., which have exhibited unique advantages, such as low production cost, high energy density, and much enhanced safety. The versatile non‐Li metal–O₂ batteries provide a better opportunity for meeting the practical requirements for sustainable energy supplies in various applications. A high‐performance cathode in non‐Li metal–O₂ batteries that can effectively trigger both oxygen reduction and evolution reactions and thus boost the overall battery performance is of great research interest. In this article, a comprehensive review on the development of Li‐free metal–O₂ batteries and particularly focusing on the oxygen catalytic cathodes for both primary and secondary non‐Li metal–O₂ batteries is carefully performed. The current challenges and potential solutions are also outlined and proposed. Through carefully selecting and rationally designing promising catalytic cathodes, a series of non‐Li metal–oxygen batteries toward practical energy storage applications are highly anticipated.     

Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications

In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast‐like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in‐growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre‐clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586–1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Mo–Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S₂MoS₂CuS₂MoS₂]^3- noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo–Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The ¹H–¹⁵N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.