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Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin.

Human cytoplasmic ferritins are heteropolymers of H and L subunits containing a catalytic ferroxidase center and a nucleation site for iron biomineralization, respectively. Here, ESI-MS successfully detected labile metal–protein interactions revealing the formation of tetra- and octa-iron clusters bound to L subunits, as previously underscored by X-ray crystallography.

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82&mdash, conserved amino acid and a key actor in cytochrome c intermolecular interactions&mdash, with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by 1H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization.

X-ray structures of homopolymeric human L-ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (μ3 -oxo)tris[(μ2 -glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the level of the protein surface, capable of contributing coordination bonds and electrostatic guidance.

Ovarian cancer represents the first cause of death for gynaecological malignancies in the western countries. Since specific and sensitive methods for early detection are missing, the large majority of patients are diagnosed at advanced stages (1). Aggressive chemotherapy with carboplatin and paclitaxel following surgery is initially efficacious, although most of the patients acquire chemoresistance and the clinical outcome in advanced ovarian cancer is very poor (1).

We investigated the kinetics of the release of iron(II) ions from the internal cavity of human H-ferritin as a function of pH. Extensive molecular dynamics simulations of the entire 24-mer ferritin provided atomic-level information on the release mechanism. Double protonation of His residues at pH 4 facilitates the removal of the iron ligands within the C3 channel through the formation of salt bridges, resulting in a significantly lower release energy barrier than pH 9.

Twenty four-mer ferritins are present in all kingdoms of life and play an essential role as iron storage proteins. The formation of a caged iron biomineral is driven by an enzymatic reaction occurring at ferroxidase centers in the central part of ferroxidase catalytically active subunits, where Fe2+ is the reaction substrate. For this purpose, Fe2+ needs to be translocated through the protein cage. Two different types of channels pierce the ferritin nanocage, in correspondence to C3 and C4 symmetry axes. The polarity across the channels controls the directional Fe2+ fluxes towards the ferroxidase centers. In vertebrate ferritins, the C3 channels have been identified as the entry ion channels coupled with the ferroxidase reaction. Here, we have prepared a series of variants of the ferritin nanocage to study the role of electrostatic residues inside the two types of channels in directioning Fe2+ substrates towards the catalytic ferroxidase sites and the inner biomineralization cavity. Interestingly, by changing the electrostatic properties of the residues at the inner edge of each channel, we can selectively activate/deactivate Fe2+ routes, modulating the rate of iron oxidation at the ferroxidase sites.

Human H and Rana catesbeiana H′ subunits in vertebrate ferritin protein cages catalyze the Fe(II) oxidation by molecular oxygen and promote the ferric oxide biomineral synthesis. By depositing iron biomineral, ferritins also prevent potentially toxic reactions products from Fe(II)-based Fenton chemistry. Recent work from our laboratory was aimed to describe the iron pathways within ferritin, from entrance into the cage to the ferroxidase site, and to understand the role played by amino-acid residues in iron trafficking and catalysis. Our approach exploits anomalous X-ray diffraction from ferritin crystals, exposed to a ferrous salt, to track transient iron binding sites along the path towards a well-defined di-iron site where they get oxidized by oxygen. Coupling structure determination with solution kinetic measurements on selected variants, allows validating the role played by key residues on the catalytic iron oxidation. Our previous studies on H′ ferritin indicated the regulatory role played by His54, and by its human counterpart Gln58, on guiding Fe(II) ions to the catalytic site. Here, we have investigated the effects induced by substituting the wild type His54 with Asn54, having different iron coordination properties. We have obtained a series of atomic-resolution crystal structures that provide time-dependent snapshots of iron bound at different locations in the H′ ferritin H54N variant. The comparison with H′ ferritin and H′ ferritin H54Q variant leads to identify a new iron binding site. Our kinetic and structural data support the role of H′ ferritin residue 54 in regulating the access of Fe(II) ions to the catalytic site.

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-L-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects., Financial support was provided by the Spanish Ministry of Economy and Competitiveness (Grants BFU2015-71017-P/BMC and BFU2015-19451/BMC, cofounded by FEDER EU), European Union (Bio-MR-00130 and CALIPSO-312284), Ramon Areces Foundation, and Andalusian Government (BIO198). B.M.-B. was awarded a PhD fellowship from the Spanish Ministry of Education (AP2009-4092) and a short-term traveling fellowship from the European Bio-NMR Project. A.G.-C. was awarded a PhD fellowship from the CSIC (JaePre-2011-01248).

Cellular uptake of human H-ferritin loaded with 50 or 350 iron ions results in significant cytotoxicity on HeLa cells at submicromolar concentrations. Conversely, Horse Spleen Ferritin, that can be considered a model of L-cages, as it contains only about 10% of H subunits, even when loaded with 1000 iron ions, is toxic only at >1 order of magnitude higher protein concentrations. We propose here that the different cytotoxicity of the two ferritin cages originates from the presence in H-ferritin of a pool of non-biomineralized iron ions bound at the ferroxidase catalytic sites of H-ferritin subunits. This iron pool is readily released during the endosomal-mediated H-ferritin internalization.

Ferritin is a ubiquitous iron concentrating nanocage protein that functions through the enzymatic oxidation of ferrous iron and the reversible synthesis of a caged ferric-oxo biomineral. Among vertebrate ferritins, the bullfrog M homopolymer ferritin is a frequent model for analyzing the role of specific amino acids in the enzymatic reaction and translocation of iron species within the protein cage. X-ray crystal structures of ferritin in the presence of metal ions have revealed His54 binding to iron(II) and other divalent cations, with its imidazole ring proposed as “gate” that influences iron movement to/from the active site. To investigate its role, His54 was mutated to Ala. The H54A ferritin variant was expressed and its reactivity studied via UV–vis stopped-flow kinetics. The H54A variant exhibited a 20% increase in the initial reaction rate of formation of ferric products with 2 or 4 Fe 2 + /subunit and higher than 200% with 20 Fe 2 + /subunit. The possible meaning of the increased efficiency of the ferritin reaction induced by this mutation is proposed taking advantage of the comparative sequence analysis of other ferritins. The data here reported are consistent with a role for His54 as a metal ion trap that maintains the correct levels of access of iron to the active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein fromRana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with itsR. catesbeianacounterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Besides its iron storage function, a new role as fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR ligand-based experiments, to provide new insights into the mechanism of ferritin/lipid interactions and the link with iron mineralization. 1D DOSY and STD NMR experiments established a stronger interaction of ferritin with unsaturated fatty acids with respect to saturated fatty acids, detergents and bile acids. Mineralization assays showed that oleate caused the most efficient increase in the initial rate of iron oxidation and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the assessed association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H chain homopolymer (HuHF), endowed with ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe2O3·H2O minerals from Fe(2+) for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe(2+) transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe(2+)/O2 catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe(2+) + O2 protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe2O3·H2O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.

Ferritins are intracellular proteins that can store thousands of iron(III) ions as a solid mineral. These structures autoassemble from four-helix bundle subunits to form a hollow sphere and are a prototypical example of protein nanocages. The protein acts as a reservoir, encapsulating iron as ferric oxide in its central cavity in a nontoxic and bioavailable form. Scientists have long known the structural details of the protein shell, owing to very high resolution X-ray structures of the apoform. However, the atomic level mechanism governing the multistep biomineralization process remained largely elusive. Through analysis of the chemical behavior of ferritin mutants, chemists have found the role of some residues in key reaction steps. Using Mössbauer and XAS, they have identified some di-iron intermediates of the catalytic reaction trapped by rapid freeze quench. However, structural information about the iron interaction sites remains scarce. The entire process is governed by a number of specific, but weak, interactions between the protein shell and the iron species moving across the cage. While this situation may constitute a major problem for crystallography, NMR spectroscopy represents an optimal tool to detect and characterize transient species involving soluble proteins. Regardless, NMR analysis of the 480 kDa ferritin represents a real challenge. Our interest in ferritin chemistry inspired us to use an original combination of solution and solid state approaches. While the highly symmetric structure of the homo-24-mer frog ferritin greatly simplifies the spectra, the large protein size hinders the efficient coherence transfer in solution, thus preventing the sequence specific assignments. In contrast, extensive (13)C-spin diffusion makes the solution (13)C-(13)C NOESY experiment our gold standard to monitor protein side chains both in the apoprotein alone and in its interaction with paramagnetic iron species, inducing line broadening on the resonances of nearby residues. We could retrieve the structural information embedded in the (13)C-(13)C NOESY due to a partial sequence specific assignment of protein backbone and side chains we obtained from solid state MAS NMR of ferritin microcrystals. We used the 59 assigned amino acids (∼33% of the total) as probes to locate paramagnetic ferric species in the protein cage. Through this approach, we could identify ferric dimers at the ferroxidase site and on their pathway towards the nanocage. Comparison with existing data on bacterioferritins and bacterial ferritins, as well as with eukaryotic ferritins loaded with various nonfunctional divalent ions, allowed us to reinterpret the available information. The resulting picture of the ferroxidase site is slightly different with various ferritins but is designed to provide multiple and generally weak iron ligands. The latter assist binding of two incoming iron(II) ions in two proximal positions to facilitate coupling with oxygen. Subsequent oxidation is accompanied by a decrease in the metal-metal distance (consistent with XAS/Mössbauer) and in the number of protein residues involved in metal coordination, facilitating the release of products as di-iron clusters under the effect of new incoming iron(II) ions.

This study summarizes results which have been obtained by a mutational study of human cytochrome c. The protein can be used as a recognition element in analytical assays and biosensors for superoxide radicals since ferricytochrome c reacts with superoxide to form ferrocytochrome c and oxygen. Here lysine mutagenesis of the distal surface (i.e., of exposed residues around the Met80 axial ligand) of human cytochrome c was pursued to evaluate the effect of the surface charges on the reaction rate with the superoxide anion radical and on the redox properties of the heme protein. The latter feature is particularly relevant when the protein is immobilized on a negatively charged self-assembled monolayer on an electrode to be used as a biosensor. The observed effects of the mutations are rationalized through structural investigations based on solution NMR spectroscopy and computational analysis of the surface electrostatics. The results suggest the presence of a specific path that guides superoxide toward an efficient reaction site. Localized positive charges at the rim of the entry channel are effective in increasing the reaction rate, whereas diffused positive charges or charges far from this area are not effective or are even detrimental, resulting in a misguided approach of the anion to the protein surface.

The use of protonless13C′–13C′ EXSY (COCO-EXSY) is proposed here to measure electron self-exchange rates. The experiment is compared to the commonly employed1H and15N EXSY experiments using as a reference system human cytochrome c. In COCO-EXSY, the exchange peaks are stronger than in the other experiments with respect to the self peaks and their intensity is less dependent on the choice of the EXSY mixing time. The use of13C directed detection may be essential for all those cases where T2relaxation is detrimental, as in the case of proteins containing highly paramagnetic metal centers, or rotating slowly in solution, or where the amide signals are difficult to detect due to chemical or conformational exchange. The proposed experiment has a general applicability and can be used to monitor exchange phenomena different from electron self-exchange.

Strong experimental evidence in animal and cellular models supports a pivotal role of sphingosine kinase-1 (SK1) in oncogenesis. In many human cancers, SK1 levels are upregulated and these increases are linked to poor prognosis in patients. Here, by employing untargeted NMR- based metabolomic profiling combined with functional validations, we report the crucial role of SK1 in the metabolic shift known as the Warburg effect in A2780 ovarian cancer cells. Indeed, expression of SK1 induced a high glycolytic rate, characterized by increased levels of lactate along with increased expression of the proton/monocarboxylate symporter MCT1, and decreased oxidative metabolism, associated with the accumulation of intermediates of the tricarboxylic acid cycle and reduction in CO2 production. Additionally, SK1-expressing cells displayed a significant increase in glucose uptake paralleled by GLUT3 transporter upregulation. The role of SK1 is not limited to the induction of aerobic glycolysis, affecting metabolic pathways that appear to support the biosynthesis of macromolecules. These findings highlight the role of SK1 signaling axis in cancer metabolic reprogramming, pointing out innovative strategies for cancer therapies.

Ferritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe(2+)-loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe(2+) uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe(2+) through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H' protein and some of its variants as case studies. We describe the structural features that determine Fe(2+) translocation with atomistic detail, and we propose a putative mechanism for Fe(2+) transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin.

Ferritins are iron-storage nanocage proteins that catalyze the oxidation of Fe2+ to Fe3+ at ferroxidase sites. By a combination of structural and spectroscopic techniques, Asp140, together with previously identified Glu57 and Glu136, is demonstrated to be an essential residue to promote the iron oxidation at the ferroxidase site. However, the presence of these three carboxylate moieties in close proximity to the catalytic centers is not essential to achieve binding of the Fe2+ substrate to the diferric ferroxidase sites with the same coordination geometries as in the wild-type cages.

The redox behavior of proteins plays a crucial part in the design of bioelectronic systems. We have demonstrated several functional systems exploiting the electron exchange properties of the redox protein cytochrome c (cyt c) in combination with enzymes and photoactive proteins. The operation is based on an effective reaction at modified electrodes but also to a large extent on the capability of self-exchange between cyt c molecules in a surface-fixed state. In this context, different variants of human cyt c have been examined here with respect to an altered heterogeneous electron transfer (ET) rate in a monolayer on electrodes as well as an enhanced self-exchange rate while being incorporated in multilayer architectures. For this purpose, mutants of the wild-type (WT) protein have been prepared to change the chemical nature of the surface contact area near the heme edge. The structural integrity of the variants has been verified by NMR and UV–vis measurements. It is shown that the single-point mutations can significantly influence the heterogeneous ET rate at thiol-modified gold electrodes and that electroactive protein/silica nanoparticle multilayers can be constructed with all forms of human cyt c prepared. The kinetic behavior of electron exchange for the mutant proteins in comparison with that of the WT has been found altered in some multilayer arrangements. Higher self-exchange rates have been found for K79A. The results demonstrate that the position of the introduced change in the charge situation of cyt c has a profound influence on the exchange behavior. In addition, the behavior of the cyt c variants in assembled multilayers is found to be rather similar to the situation of cyt c self-exchange in solution verified by NMR.

Erratum in : J Biol Chem. 2013 Jan 25;288(4):2190.; International audience; The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Molecular size has limited solution NMR analyses of proteins. We report (13)C-(13)C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting (13)C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C-C connectivities between nuclei separated by 1-4 bonds. These results show the potential of (13)C-(13)C NOESY for solution studies of molecular assemblies100 kDa.

We provide here an historical perspective of NMR applied to iron-containing proteins. At first, the field developed using paramagnetic NMR: the 1H NMR spectra of heme and FeS proteins were used as clear spectroscopic fingerprints of the electronic structure of the metal ion and its inner and outer coordination spheres. Starting 1994, NMR of metalloproteins was focused more on the protein part and the determination of the 3D structures in solution of small proteins was achieved; paramagnetic NMR observables were exploited as non-conventional NMR constraints. With time, NMR has gained attention as a methodology to monitor protein–protein interactions, becoming a unique tool to learn about the interaction surfaces in weak transient complexes. The recent interest in the understanding of metal ion homeostatis and metal cofactor assembly has led to a renewed interest in the application of NMR: (i) as a spectroscopic tool, to characterize the novel binding site of metal centers where the metal is no more an integral part of the protein but rather a chemical entity that has to be transferred along trafficking pathways or a cofactor that has to undergo specific reactions and (ii) as a structural method for the definition of the metal-mediated protein–protein interactions. The reported case examples cover the bacterial heme acquisition system, the cytosolic FeS protein assembly and mitochondrial FeS cluster assembly machineries and the eukaryotic iron-storage ferritin.

S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metal-deficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior.

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.

Recent NMR structural and dynamical data on partially folded forms of mono-heme cytochrome c provide a unifying picture of the behavior of the protein far from the native conditions and suggest useful hints to explain the redox dependent stability of the protein. A fragile hinge in the structure of mitochondrial cytochrome c is identified, which may not have correspondents in smaller type-1 cytochromes. Former spectroscopic and kinetic data are here discussed in terms of this new view.

The reversible unfolding of oxidized Bacillus pasteurii cytochrome c(553) by guanidinium chloride under equilibrium conditions has been monitored by NMR and optical spectroscopy. The results obtained indicate that unfolding takes place through a mechanism involving the detachment from heme iron coordination of the sulfur of the Met71 axial ligand and yielding either a high spin (HS) or a low spin (LS(1)) species, depending on the pH value. In the LS(1) form the Met71 is replaced by another protein ligand, possibly Lys. The ligand exchange reaction does not reach completion until the protein backbone reaches a largely unfolded state, as monitored through 1H-15N NMR experiments, thus demonstrating that there is a significant correlation between formation of the Fe-S bond and native structure stability. 1H/2H exchange data, however, show that helix alpha(3), the C-terminal region of helix alpha(4), and helix alpha(5) maintain low exchangeability of the amide protons in the LS(1) form. This finding most likely implies that these regions maintain some ordered non-covalent structure, in which the amide moieties are involved in H-bonds. Finally, a folding mechanism is proposed and discussed in terms of analogies and differences with the larger mitochondrial cytochrome c proteins. It is concluded that the thermodynamic stability of the region around the metal cofactor is determined by the chemical nature of the residues around the axial methionine residue.

Lysines 9 and 10 in Desulfuromonas acetoxidans cytochrome c7, which could be involved in the interaction mechanism with the redox partners, have been replaced by alanine residues using site-directed mutagenesis. The solution structure of the fully oxidized form of K9-10A cytochrome c7, which is paramagnetic with three paramagnetic centers, has been determined via 1H NMR. The assignment of the spectra has been performed through an automatic program whose algorithm and strategy are here described. The assignment of the NOESY spectra has been further extended by back calculating the NOESY maps. The final number of meaningful NOE-based upper distance limits was 1186. In the Restrained Energy Minimization calculations, 147 pseudocontact shift constraints were also included, which showed consistency with NOE-based constraints and therefore further contribute to validate the structure quality. A final family of 35 conformers was calculated with RMSD values with respect to the mean structure of 0.69 ± 0.17 A and 1.05 ± 0.14 A for the backbone and heavy atoms, respectively. The overall fold of the molecule is maintained with respect to the native protein. The loop present between heme III and heme IV results to be highly disordered also in the present structure although its overall shape mainly resembles that of the oxidized native protein, and the two strands which give rise to the short β-sheet present at the N-terminus and connected by a turn containing the mutated residues, are less clearly defined. If this loop is neglected, the RMSD values are 0.52 ± 0.07 A and 0.92 ± 0.06 A for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The relative distances and orientations of the three hemes are maintained, as well as the orientation of the imidazole rings of the axial histidine ligands, with the only exception of heme IV. Such difference probably reflects minor conformational changes due to the substitution of the vicinal Lys10 with an Ala. The replacement of the two lysines does not affect the reduction potentials of the three hemes, consistently with the expectations on the basis of the structure and electrostatic calculations. However, the replacement of the two lysines affects the reactivity of the mutant cytochrome c7 with [Fe] hydrogenase, inducing a change in Km. This finding is in agreement with the identification of the protein area around heme IV as the interacting site.

Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages.

Immunoelectron microscopy analysis of brain tissue sections and rat-specific sandwich ELISA allowed the localization of interleukin-1beta (IL-1beta) immunoreactivity in the mitochondria and cytosol of neocortical tissue preparations from the brain of naive, untreated, rats and rats receiving a single daily injection into one lateral cerebral ventricle (i.c.v.) of bovine serum albumin (BSA; 100 ng/day) for seven consecutive days. Interestingly, seven days i.c.v. treatment with the HIV-1 coat protein gp120 (100 ng/day) enhances IL-1beta immunoreactivity in the cellular fractions studied. Elevation of mitochondrial immunoreactive IL-1beta levels seems to originate from the conversion operated by the interleukin converting enzyme (ICE) of mitochondrial pro-IL-1beta; in fact, IL-1beta increases reported in the ELISA experiments were paralleled by a decrease of the mitochondrial pro-IL-1beta 31-kDa band in conjunction with enhanced expression of the p20 component of activated ICE. In conclusion, the present results demonstrate that gp120-enhanced neocortical expression of IL-1beta originates, at least in part, from in situ cleavage of mitochondrial pro-IL-1beta and suggest that this, together with the central role of the mitochondrion in the expression of programmed cell death, may be important for apoptosis induced by the viral coat protein in the brain of rats.

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.

The solution structure of Desulfuromonas acetoxidans cytochrome c7 has been refined by using 1H-NMR spectra recorded at 800 MHz and by using pseudocontact shifts in the final energy minimization procedure. The protein, composed of 68 amino acids, contains three paramagnetic heme moieties, each with one unpaired electron. The largely distributed paramagnetism broadens the lines in several protein parts. The structure is now relatively well resolved all over the backbone by the use of 1315 meaningful NOEs and 90 pseudocontact shifts. The statistical analysis of the structure indicates its satisfactory quality. The protein-fold is quite similar to that of the analogous four-heme cytochromes c3 for those parts which can be considered homologous. The solvent accessibility and the electrostatic potential surfaces surrounding the three hemes have been analyzed in terms of their reduction potentials. The resulting magnetic susceptibility anisotropy data obtained from pseudocontact shifts are analyzed in terms of structural data.

Molecular dynamics calculations have been performed over long trajectories with the inclusion of explicit solvent molecules on the reduced and the oxidized states of yeast iso-1-cytochrome c. The resulting structures have been analyzed and compared both in terms of structural properties and dynamical behavior. The structure of the buried water molecules around the heme has been also analyzed for the two oxidation states and compared with the experimental observations on the X-ray and the solution NMR structures. From the overall analysis we learn that, as also observed experimentally through NMR, no significant differences are present between the structures of the two oxidation states beside the arrangement of a few side chains. Also the internal mobility is similar for the two oxidation states, even if interesting differences are observed for some residues, as for Tyr67, a residue present at the heme site. The location and the mobility of the ordered water molecules, observed in solution by NMR, are completely reproduced in the molecular dynamics simulations, which have been able to predict the different displacements of the catalytically relevant water molecule WAT166, similar to those observed in solution for the two oxidation states, at variance with that observed in the starting crystallographic structures. The relevance of these findings with respect to the prediction of structural and dynamical properties is discussed.

The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only.

Background: Cytochrome c has five distinct pH-dependent conformational states, including two alkaline forms of unknown structure. It is believed that in both of the alkaline forms a Lys residue is ligated to the heme, but the identity of the Lys residue is different. Exchange between these forms would require extensive structural rearrangement. Mutation of the heme axial ligand (Met80) to Ala in Saccharomyces cerevisiae iso-l-cytochrome c yields a protein (Ala80cyt c ) capable of binding exogenous ligands such as dioxygen and cyanide. We have analyzed the 1 H NMR spectra of this mutant at various pH values in the hope of gaining insight into the structure of the acidic and alkaline forms of native cytochrome c . Results: The pH dependence of the 1 H NMR spectrum of ferriAla80cyt c is consistent with the high-spin/low-spin transition (pK a = 6.5) observed by absorption spectroscopy. The T 1 values for the low-spin form are consistent with OH − ligation, as inferred previously from absorption and electron paramagnetic resonance spectroscopic results. The pH-dependent equilibria of ferriAla80cyt c differ from those of the wild-type protein. Both Ala80 and wild-type ferricyt c appear to have the same iron coordination at low pH (≈ 2), while only one alkaline form of Ala80cyt c (versus two for WTcyt c ) was detected. Conclusions: The differences between the pH dependence of the 1 H NMR spectra of Ala80cyt c and those of the wild-type protein demonstrate that the heme axial ligands influence the relative energies of the conformational states of cytochrome c . The results are consistent with the notion that a large rearrangement is required to switch between the two alkaline forms.

The first step of iron biomineralization mediated by ferritin is the oxidation at the ferroxidase active site of two ferrous ions to a diferric oxo/hydroxo species. Metal-loaded ferritin crystals obtained by soaking crystals of frog ferritin in FeSO(4) and CuSO(4) solutions followed by flash freezing provided X-ray crystal structures of the tripositive iron and bipositive copper adducts at 2.7 and 2.8 A resolution, respectively. At variance with the already available structures, the crystal form used in this study contains 24 independent subunits in the asymmetric unit permitting comparison between them. For the first time, the diferric species at the ferroxidase site is identified in ferritins from higher eukaryotes. Anomalous difference Fourier maps for crystals (iron crystal 1) obtained after long soaking times in FeSO(4) solution invariantly showed diferric species with a Fe-Fe average distance of 3.1 ± 0.1 A, strongly indicative of the presence of a μ-oxo/hydroxo bridge between the irons; protein ligands for each iron ion (Fe1 and Fe2) were also unequivocally identified and found to be the same in all subunits. For copper bound ferritin, dicopper(II) centers are also observed. While copper at site 1 is essentially in the same position and has the same coordination environment as Fe1, copper at site 2 is displaced toward His54, now acting as a ligand; this results in an increased intermetal distance (4.3 ± 0.4 A). His54 coordination and longer metal-metal distances might represent peculiar features of divalent cations at the ferroxidase site. This oxidation-dependent structural information may provide key features for the mechanistic pathway in ferritins from higher eukaryotes that drive uptake of bivalent cation and release of ferric products at the catalytic site. This mechanism is supported by the X-ray picture obtained after only 1 min of soaking in FeSO(4) solutions (iron crystal 2) which reasonably contain the metal at different oxidation states. Here two different di-iron species are trapped in the active site, with intermetal distances corresponding to those of the ferric dimer in crystal 1 and of the dicopper centers and corresponding rearrangement of the His54 side chain.

Here, we report on cytochrome c/bilirubin oxidase multilayer electrodes with different cytochrome c (cyt c) forms including mutant forms of human cyt c, which exhibit different reaction rates with bilirubin oxidase (BOD) in solution. The multilayer formation via the layer-by-layer technique and the kinetic behavior of the mono (only cyt c) and biprotein (cyt c and BOD) multilayer systems are studied by SPR and cyclic voltammetry. For the layer construction, sulfonated polyaniline is used. The only cyt c containing multilayer electrodes show that the quantity of deposited protein and the kinetic behavior depend on the cyt c form incorporated. In the case of the biprotein multilayer with BOD, it is demonstrated that the catalytic signal chain from the electrode via cyt c to BOD and oxygen can be established with all chosen cyt c forms. However, the magnitude of the catalytic current as well as the kinetic behavior differ significantly. We conclude that the different cytochrome c forms affect three parameters, identified here, to be important for the functionality of the multilayer system: the amount of molecules per layer, which can be immobilized on the electrodes, the cyt c self-exchange rate, and the rate constant for the reaction with BOD.

International audience; A structural model of the adduct between human cytochrome c and the human anti-apoptotic protein Bcl-x(L), which defines the protein-protein interaction surface, was obtained from solution NMR chemical shift perturbation data. The atomic level information reveals key intermolecular contacts identifying new potentially druggable areas on cytochrome c and Bcl-x(L). Involvement of residues on cytochrome c other than those in its complexes with electron transfer partners is apparent. Key differences in the contact area also exist between the Bcl-x(L) adduct with the Bak peptide and that with cytochrome c. The present model provides insights to the mechanism by which cytochrome c translocated to cytosol can be intercepted, so that the apoptosome is not assembled.

The 1H NMR spectroscopy was used to study lignin peroxidase (LiP) and manganese peroxidase (MnP) containing deuterated histidines. LiP and MnP were obtained from a histidine auxotroph of the fungus Phanerochaete chrysosporium grown in the presence of deuterated histidines. The derivatives with deuterated histidines have allowed a firm assignment of the protons of the distal and proximal histidines. We have also found that the LiP from this strain exhibits different orientations of the 2-vinyl group compared to the LiP from the strain previously studied. Mobility of the group has also been detected, thus explaining the apparent inconsistency between X-ray solid-state and NMR solution data. The 15N shift values of 15N-enriched CN- in the cyanide derivatives of LiP and MnP have also been measured. The shift patterns, both for 15N and for the proximal histidine protons of several peroxidases, are consistent with predominant contact shift contributions which reflect the bond strength of the metal-axial ligand. Finally, our results confirm a correlation between shift values of 15N and those of proximal histidine protons and the Fe3+/Fe2+ redox potentials.

The possibilities and the limitations of 2D NMR for the structural characterization of paramagnetic metalloproteins are reviewed. We survey the general strategies for 2D1H NMR investigations of hyperfine shifted signals. Careful adaptation of classical 2D NMR experiments to fast relaxing systems results in the detection of previously not observed scalar and dipolar connectivities, thus leading to the specific assignment of selected resonances. The approach is of general applicability for paramagnetic metalloproteins. We report here on the application of the application of the method to an iron sulfur protein and a heme protein. In both cases specific assignment of several hyperfine shifted signals, corresponding to active site protons, were obtained; this allowed significant insight into the structure-function relationships of these metalloproteins.

1H NMR spectra at 200- and 600-MHz of manganese peroxidase from Phanerochaete chrysosporium and of its cyanide derivative are reported. The spectrum of the native protein is very similar to that of other peroxidases. The assignment of the spectrum of the cyanide derivative has been performed through 1D NOE, 2D NOESY, and COSY experiments. This protein is very similar to lignin peroxidase, the only meaningful difference being the shift of H delta 2 of the proximal histidine. The spectra of the cyanide derivative of these two proteins are compared with those of horseradish peroxidase and cytochrome c peroxidase. The shift pattern of the protons of the proximal histidine is discussed relative to the structural properties which affect the Fe3+/Fe2+ redox potential.

Ferritin is a multimeric nanocage protein that directs the reversible biomineralization of iron. At the catalytic ferroxidase site two iron(II) ions react with dioxygen to form diferric species. In order to study the pathway of iron(III) from the ferroxidase site to the central cavity a new NMR strategy was developed to manage the investigation of a system composed of 24 monomers of 20 kDa each. The strategy is based on 13 C- 13 C solution NOESY experiments combined with solid-state proton-driven 13 C- 13 C spin diffusion and 3D coherence transfer experiments. In this way, 75% of amino acids were recognized and 35% sequence-specific assigned. Paramagnetic broadening, induced by iron(III) species in solution 13 C- 13 C NOESY spectra, localized the iron within each subunit and traced the progression to the central cavity. Eight iron ions fill the 20-Å-long iron channel from the ferrous/dioxygen oxidoreductase site to the exit into the cavity, inside the four-helix bundle of each subunit, contrasting with short paths in models. Magnetic susceptibility data support the formation of ferric multimers in the iron channels. Multiple iron channel exits are near enough to facilitate high concentration of iron that can mineralize in the ferritin cavity, illustrating advantages of the multisubunit cage structure.

The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni(2+) for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni(2+) insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni(2+) (Kd=0.15 microM) or Zn(2+) (Kd=0.49 microM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn(2+) and Ni(2+), which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE-HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn(2+), and not for Ni(2+), in the stabilization of this complex was demonstrated using size-exclusion chromatography, light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of a novel binding site for Zn(2+), actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn(2+) in the Ni(2+)-dependent urease system is envisaged.

The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV-vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.