Blue copper proteins: a comparative analysis of their molecular interaction properties

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.     

NMR of Redox Proteins of Plants, Yeasts and Photosynthetic Bacteria

NMR spectroscopy has evolved dramatically over the past 15 years, establishing a new, reliable methodology for studying biomacromolecules at atomic resolution. The three-dimensional structure and dynamics of a biomolecule or a biomolecular complex is only one of the main types of information available using NMR. The spectral assignment to the specific nuclei of a biostructure is a very precise reflection of their electronic environment. Any change in this environment due to a structural change, the binding of a ligand or the redox state of a redox cofactor, will be very sensitively reported by changes in the different NMR parameters. The capabilities of the NMR method are currently expanding dramatically and it is turning into a powerful means to study biosystems dynamically in exchange between different conformations, exchanging ligands, transient complexes, or the activation/inhibition of regulated enzymes. We review here several NMR studies that have appeared during the past 5 or 6 years in the field of redox proteins of plants, yeasts and photosynthetic bacteria. These new results illustrate the recent biomolecular NMR evolution and provide new physiological models for understanding the different types of electron transfer, including glutaredoxins, thioredoxins and their dependent enzymes, the ferredoxin-NADP oxidoreductase complex, flavodoxins, the plastocyanin-cytochrome f complex, and cytochromes c.     

Predicting the redox state and secondary structure of cysteine residues in proteins using NMR chemical shifts

Different programs and methods were employed to superimpose protein structures, using members of four very different protein families as test subjects, and the results of these efforts were compared. Algorithms based on human identification of key amino acid residues on which to base the superpositions were nearly always more successful than programs that used automated techniques to identify key residues. Among those programs automatically identifying key residues, MASS could not superimpose all members of some families, but was very efficient with other families. MODELLER, MultiProt, and STAMP had varying levels of success. A genetic algorithm program written for this project did not improve superpositions when results from neighbor-joining and pseudostar algorithms were used as its starting cases, but it always improved superpositions obained by MODELLER and STAMP. A program entitled PyMSS is presented that includes three superposition algorithms featuring human interaction.     

S100-annexin complexes: some insights from structural studies

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex.     

Molecular basis for redox-Bohr and cooperative effects in cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774: crystallographic and modeling studies of oxidized and reduced high-resolution structures at pH 7.6

The tetraheme cytochrome c3 is a small metalloprotein with ca. 13,000 Da found in sulfate-reducing bacteria, which is believed to act as a partner of hydrogenase. The three-dimensional structure of the oxidized and reduced forms of cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 at pH 7.6 were determined using high-resolution X-ray crystallography and were compared with the previously determined oxidized form at pH 4.0. Theoretical calculations were performed with both structures, using continuum electrostatic calculations and Monte Carlo sampling of protonation and redox states, in order to understand the molecular basis of the redox-Bohr and cooperativity effects related to the coupled transfer of electrons and protons. We were able to identify groups that showed redox-linked conformational changes. In particular, Glu61, His76, and propionate D of heme II showed important contributions to the redox-cooperativity, whereas His76, propionate A of heme I, and propionate D of heme IV were the key residues for the redox-Bohr effect. Upon reduction, an important movement of the backbone region surrounding hemes I and II was also identified, that, together with a few redox-linked conformational changes in side-chain residues, results in a significant decrease in the solvent accessibility of hemes I and II.     

A hypothetical complex between crystalline flavocytochrome b2 and Cytochrome c

Flavocytochrome b₂ and cytochrome c are physiological electron transfer partners in yeast mitochondria. The formation of a stable complex between them has been demonstrated both in solution and in the crystalline state. On the basis of the three-dimensional structures, using molecular modeling and energy minimization, we have generated a hypothetical model for the interaction of these redox partners in the crystal lattice. General criteria such as good charge and surface complementarity, plausible orientation, and separation distance of the prosthetic groups, as well as more specific criteria such as the stoichiometry determined in the crystal, and the involvement of both domains and of more than one subunit of flavocytochrome b₂ led us to discriminate between several possible interaction sites. In the hypothetical model we present, four cytochrome c molecules interact with a tetramer of flavocytochrome b₂. The b₂ and c hemes are coplanar, with an edge-to-edge distance of 14 Å. the contact surface area is ca. 800 Ų. Several electrostatic interactions involving the flavin and the heme domains of flavocytochrome b₂ stabilize the binding of cytochrome c. © 1993 Wiley-Liss, Inc.     

The dynamic duo: Combining NMR and small angle scattering in structural biology

Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X‐ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well‐suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography, and SAS is, thus, very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, SAS provides additional structural information and can differentiate between NMR‐derived models. Scattering data can also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state‐of‐the‐art approaches for combining NMR, crystallography, and SAS data to characterize protein complexes in solution.     

S-glutathionylation: from redox regulation of protein functions to human diseases

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an integral role in the modulation of several physiological functions but can also be potentially destructive if produced in excessive amounts. Protein cysteinyl thiols appear especially sensitive to ROS/RNS attack. Experimental evidence started to accumulate recently, documenting that S-glutathionylation occurs in a number of physiologically relevant situations, where it can produce discrete modulatory effects on protein function. The increasing evidence of functional changes resulting from this modification, and the growing number of proteins shown to be S-glutathionylated both in vitro and in vivo support this contention, and confirm this as an attractive area of research. S-glutathionylated proteins are now actively investigated with reference to problems of biological interest and as possible biomarkers of human diseases associated with oxidative/nitrosative stress.     

Role of mediators in electron transport from glucose oxidase redox centre to electrode surface in a covalently coupled enzyme paste electrode

We have studied the glucose oxidase immobilized carbon paste electrodes in the presence and absence of small mediator molecules. We have used p-benzoquinone and riboflavin as mediators in our studies. The effect of mediator molecules on the electron transfer between the enzyme redox centre and the electrode surface was explained from the cyclic voltammograms and rotating disk electrode data. In the absence of oxygen, we have noted that the mediators play a central role in the electron transfer. We have also proposed a possible mechanism for the electron transfer from enzyme active site to the electrode surface via mediators, based on our observations. Dedicated to the memory of Professor J Das     

Resuspension studies in cylindrical microcosms: Effects of stirring velocity on the dynamics of redox sensitive elements in a coastal sediment

Effects of resuspension on the release of dissolved, redox sensitive elements (Fe, Mn) was studied in cylindrical microcosms. Effects from changing water stirring velocity in sediment pools were evaluated through measurements of pore water profiles of dissolved Mn, Fe and redox potential. Mn was a good natural marker to follow such effects. At current velocities below the threshold velocity for resuspension (37 cm s-1), Mn release rates to overlying water were 100 times higher compared to steady-state values. Pulse increases in Mn concentration were the result of convective currents inside flow chambers. These results were strongly supported by measurements of Eh profiles in the sediment pore water. Furthermore, impacts from increasing stirring velocity were found down to 1.9 cm depth below the resuspended layer of sediment.     

Lipid nanotechnologies for structural studies of membrane‐associated proteins

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane‐associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo‐electron microscopy (cryo‐EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ～20 nm inner diameter and a few microns in length, that self‐assemble in aqueous solutions. The lipid nanodisks (NDs) are self‐assembled discoid lipid bilayers of ～10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane‐associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane‐bound coagulation factor VIII in vitro for structure determination by cryo‐EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three‐dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane‐associated proteins and complexes for structural studies by cryo‐EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane‐associated proteins, such as the coagulation factors, at a close to physiological environment. Proteins 2014; 82:2902–2909. © 2014 Wiley Periodicals, Inc.     

NMR studies of electron carrier proteins from sulphate reducing bacteria.

The sulphate-reducing bacteria have a complex electron transfer system which leads to the reduction of sulphate by oxidation of either organic substrates or molecular hydrogen. These bacteria can either produce or consume molecular hydrogen. The central part of this electron pathway for Desulovibrio gigas is constituted by hydrogenase (3 X (4Fe-4S)). cytochrome c3 (4 haems with different redox potentials) and a one (4Fe-4S) cluster ferredoxin. This ferredoxin is isolated in different oligomeric forms, which stabilize different oxidation states and have different physiological roles; the trimer FdI being involved in the production of H2 and the tetramer FdII being more efficient for the consumption of H2. The presence of intrinsic probes (the iron ions) in these proteins is particularly helpful for structural studies using NMR spectroscopy. These studies allowed a characterization of the oxidation states used by the different oligomers of the ferredoxin and obtaintion of structural information on multi-haem cytochromes (c3 and c7). NMR is also suitable to study protein-protein interaction. The study of the complex formed between FdII and cytochrome c3 has shown that there is an alteration of the kinetics of electron transfer upon complexation.     

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D ¹H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D ¹H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R _2p of the resonances can be determined, and the conditions kR _2p or _pkR _2p are fulfilled, _p being the frequency separation between corresponding diamagnetic and paramagnetic sites.     

Microscale purification of proteins by line immunoelectrophoresis: Application of the technique in protein biogenesis studies

A small-scale version of line immunoelectrophoresis in combination with immunoprecipitate excision is describeb as a rapid convenient technique to purify proteins on a micro scale in biogenesis studies. In the purification of pig small intestinal microvillar enzymes the method was found to be capable of a quantitative purification and to result in a higher state of purity than an isolation procedure using protein A-Sepharose. Since the method furthermore allows a simultaneous purification of several different protein antigens from the sample, it may be of interest as an alternative method to other procedures in the purification of proteins on a micro scale.     

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.     

Production of small cysteine‐rich effector proteins in Escherichia coli for structural and functional studies

Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which aid in microbial infection. In the case of eukaryotic filamentous pathogens, such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here, we present a general scheme and methodology for the production and characterization of small cysteine‐rich (SCR) effectors utilizing Escherichia coli expression systems. Using this approach, we successfully produced cysteine‐rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallization and functional experiments. These results are discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest.     

Nitric oxide reduction in BioDeNOx reactors: kinetics and mechanism

Biological reduction of nitric oxide (NO) to di-nitrogen (N(2)) gas in aqueous Fe(II)EDTA(2-) solutions is a key reaction in BioDeNOx, a novel process for NOx removal from flue gases. The mechanism and kinetics of the first step of NO reduction, that is, the conversion of NO to N(2)O, was determined in batch experiments using various types of inocula. Experiments were performed in Fe(II)EDTA(2-) medium (5-25 mM) under BioDeNOx reactor conditions (55 degrees C, pH 7.2 +/- 0.2) with ethanol as external electron donor. BioDeNOx reactor mixed liquor gave the highest NO reduction rates (+/-0.34 nmol s(-1) mg(prot)(-1)) with an estimated K(m) value for NO lower than 10 nM. The specific NO (to N(2)O) reduction rate depended on the NO (aq) and Fe(II)EDTA(2-) concentration as well as the temperature. The experimental results, complemented with kinetic and thermodynamic considerations, show that Fe(II)EDTA(2-), and not ethanol, is the primary electron donor for NO reduction, that is, the BioDeNOx reactor medium (the redox system Fe(II)EDTA(2-)/Fe(III)EDTA(-)) interferes with the NO reduction electron transfer chain and thus enhances the NO denitrification rate.     

Extraction of detergents from hydrophobic proteins with isopentanol: application to electrophoretic analysis of photosynthetic bacterial hydrophobic proteins

The method for extracting Triton X-100 used by I. H. Mather and C. B. Tampling [Anal. Biochem. 93, 139-142 (1979)], has been extended to other detergents of different charge and chemical nature. All the detergents tested can be extracted with isopentanol in conditions in which not more than 8% of hydrophobic or hydrophilic protein is lost from the water phase. The removal of detergent from reaction centers and light harvesting protein-pigment complexes of photosynthetic bacteria, eliminates the artifacts of oligomers when analyzed by sodium dodecyl sulfate-gel electrophoresis.     

Thermodynamic Redox Properties Governing the Half-Reduction Characteristics of Histamine Dehydrogenase from Nocardioides simplex

Histamine dehydrogenase from Nocardioides simplex is a homodimer and belongs to the family of iron-sulfur flavoproteins having one [4Fe-4S] cluster and one 6-S-cysteinyl FMN per monomer. In the reductive titration with histamine, two-electron reduction occurred per monomer at pH<9, while single-electron reduction proceeded at pH>9. The substrate-reduced histamine dehydrogenase yielded an electron paramagnetic resonance spectral signal assigned to the flavin semiquinone. The signal intensity increased with pH up to pH 9 and reached a maximum at pH>9. These unique features are explained in terms of the redox potential of the cofactors, where the redox potential was evaluated over a pH range from 7 to 10 by using a spectroelectrochemical titration method for the flavin and cyclic voltammetry for the [4Fe-4S] cluster. The bell-type pH dependence of the enzymatic activity is also discussed in terms of the pH dependence of the centers’ redox potential.     

Iron-sulfur flavoenzymes: the added value of making the most ancient redox cofactors and the versatile flavins work together

Iron-sulfur (Fe-S) flavoproteins form a broad and growing class of complex, multi-domain and often multi-subunit proteins coupling the most ancient cofactors (the Fe-S clusters) and the most versatile coenzymes (the flavin coenzymes, FMN and FAD). These enzymes catalyse oxidoreduction reactions usually acting as switches between donors of electron pairs and acceptors of single electrons, and vice versa. Through selected examples, the enzymes'' structure−function relationships with respect to rate and directionality of the electron transfer steps, the role of the apoprotein and its dynamics in modulating the electron transfer process will be discussed.