Cupredoxins--a study of how proteins may evolve to use metals for bioenergetic processes.
ABSTRACT: Cupredoxins are small proteins that contain type I copper centers, which are ubiquitous in nature. They function as electron transfer shuttles between proteins. This review of the structure and properties of native cupredoxins, and those modified by site-directed mutagenesis, illustrates how these proteins may have evolved to specifically bind copper, develop recognition sites for specific redox partners, tune redox potential for a particular function, and allow for efficient electron transfer through the protein matrix. This is relevant to the general understanding of the roles of metals in energy metabolism, respiration and photosynthesis.
Project description:Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.
Project description:Cupredoxins are widespread copper-binding proteins, mainly involved in electron transfer pathways. They display a typical rigid greek key motif consisting of an eight stranded β-sandwich. A fascinating feature of cupredoxins is the natural diversity of their copper center geometry. These geometry variations give rise to drastic changes in their color, such as blue, green, red or purple. Based on several spectroscopic and structural analyses, a connection between the geometry of their copper-binding site and their color has been proposed. However, little is known about the relationship between such diversity of copper center geometry in cupredoxins and possible implications for function. This has been difficult to assess, as only a few naturally occurring green and red copper sites have been described so far. We report herein the spectrocopic characterization of a novel kind of single domain cupredoxin of green color, involved in a respiratory pathway of the acidophilic organism Acidithiobacillus ferrooxidans. Biochemical and spectroscopic characterization coupled to bioinformatics analysis reveal the existence of some unusual features for this novel member of the green cupredoxin sub-family. This protein has the highest redox potential reported to date for a green-type cupredoxin. It has a constrained green copper site insensitive to pH or temperature variations. It is a green-type cupredoxin found for the first time in a respiratory pathway. These unique properties might be explained by a region of unknown function never found in other cupredoxins, and by an unusual length of the loop between the second and the fourth copper ligands. These discoveries will impact our knowledge on non-engineered green copper sites, whose involvement in respiratory chains seems more widespread than initially thought.
Project description:Cupredoxins are electron-transfer proteins that have active sites containing a mononuclear Cu center with an unusual trigonal monopyramidal structure (Type 1 Cu). A single Cu-Scys bond is present within the trigonal plane that is responsible for its unique physical properties. We demonstrate that a cysteine-containing variant of streptavidin (Sav) can serve as a protein host to model the structure and properties of Type 1 Cu sites. A series of artificial Cu proteins are described that rely on Sav and a series of biotinylated synthetic Cu complexes. Optical and EPR measurements highlight the presence of a Cu-Scys bond, and XRD analysis provides structural evidence. We further provide evidence that changes in the linker between the biotin and Cu complex within the synthetic constructs allows for small changes in the placement of Cu centers within Sav that have dramatic effects on the structural and physical properties of the resulting artificial metalloproteins. These findings highlight the utility of the biotin-Sav technology as an approach for simulating active sites of metalloproteins.
Project description:Protein-mediated electron transfer is an essential event in many biochemical processes. Efficient electron transfer requires the reorganization energy of the redox event to be minimized, which is ensured by the presence of rigid donor and acceptor sites. Electron transfer copper sites are present in the ubiquitous cupredoxin fold, able to bind one or two copper ions. The low reorganization energy in these metal centers has been accounted for by assuming that the protein scaffold creates an entatic/rack-induced state, which gives rise to a rigid environment by means of a preformed metal chelating site. However, this notion is incompatible with the need for an exposed metal-binding site and protein-protein interactions enabling metallochaperone-mediated assembly of the copper site. Here we report an NMR study that reveals a high degree of structural heterogeneity in the metal-binding region of the nonmetallated Cu(A)-binding cupredoxin domain, arising from microsecond to second dynamics that are quenched upon metal binding. We also report similar dynamic features in apo-azurin, a paradigmatic blue copper protein, suggesting a general behavior. These findings reveal that the entatic/rack-induced state, governing the features of the metal center in the copper-loaded protein, does not require a preformed metal-binding site. Instead, metal binding is a major contributor to the rigidity of electron transfer copper centers. These results reconcile the seemingly contradictory requirements of a rigid, occluded center for electron transfer, and an accessible, dynamic site required for in vivo copper uptake.
Project description:Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key ?-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, ?3DChC2. (1) While this design demonstrated that a ?-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide ?3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRAND?3D (GR?3D) with ? Gu = -11.4 kcal/mol compared to the original design's -5.1 kcal/mol. Diffraction quality crystals were grown of GR?3D (a first for an ?3D peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GR?3DChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs' hyperfine coupling constant from 160 to 127 × 10-4 cm-1. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41-Cu interaction leads to the ?3DChC2 construct's red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.
Project description:The C-terminal copper-binding loop in the beta-barrel fold of the cupredoxin azurin has been replaced with a range of sequences containing alanine, glycine, and valine residues to assess the importance of amino acid composition and the length of this region. The introduction of 2 and 4 alanines between the coordinating Cys, His, and Met results in loop structures matching those in naturally occurring proteins with the same loop lengths. A loop with 4 alanines between the Cys and His and 3 between the His and Met ligands has a structure identical to that of the WT protein, whose loop is the same length. Loop structure is dictated by length and not sequence allowing the properties of the main surface patch for interactions with partners, to which the loop is a major contributor, to be optimized. Loops with 2 amino acids between the ligands using glycine, alanine, and valine residues have been compared. An empirical relationship is found between copper site protection by the loop and reduction potential. A loop adorned with 4 methyl groups is sufficient to protect the copper ion, enabling most sequences to adequately perform this task. The mutant with 3 alanine residues between the ligands forms a strand-swapped dimer in the crystal structure, an arrangement that has not, to our knowledge, been seen previously for this family of proteins. Cupredoxins function as redox shuttles and are required to be monomeric; therefore, none have evolved with a metal-binding loop of this length.
Project description:Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read-out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter-copper electron transfer catalyzed by CuNiRs. Cryoreduction-EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox-center containing proteins (homo and hetero metal ions).
Project description:A well-defined copper complex bearing iminosemiquinone ligands performs single electron reduction of an electrophilic CF3+ source into CF?3 radicals. This redox behavior is enabled by the ligand which shuttles through two different redox states (iminosemiquinone and iminobenzoquinone) while the copper center is preserved as a Cu(ii). This system was used in the trifluoromethylation of silyl enol ethers, heteroaromatics and in the hydrotrifluoromethylation of alkynes. This is the first example of cooperative redox catalysis for the controlled generation of CF?3 radicals.
Project description:The current understanding of dissimilatory metal reduction is based primarily on isolates from the proteobacterial genera Geobacter and Shewanella. However, environments undergoing active Fe(III) reduction often harbor less-well-studied phyla that are equally abundant. In this work, electrochemical techniques were used to analyze respiratory electron transfer by the only known Fe(III)-reducing representative of the Acidobacteria, Geothrix fermentans. In contrast to previously characterized metal-reducing bacteria, which typically reach maximal rates of respiration at electron acceptor potentials of 0 V versus standard hydrogen electrode (SHE), G. fermentans required potentials as high as 0.55 V to respire at its maximum rate. In addition, G. fermentans secreted two different soluble redox-active electron shuttles with separate redox potentials (-0.2 V and 0.3 V). The compound with the lower midpoint potential, responsible for 20 to 30% of electron transfer activity, was riboflavin. The behavior of the higher-potential compound was consistent with hydrophilic UV-fluorescent molecules previously found in G. fermentans supernatants. Both electron shuttles were also produced when cultures were grown with Fe(III), but not when fumarate was the electron acceptor. This study reveals that Geothrix is able to take advantage of higher-redox-potential environments, demonstrates that secretion of flavin-based shuttles is not confined to Shewanella, and points to the existence of high-potential-redox-active compounds involved in extracellular electron transfer. Based on differences between the respiratory strategies of Geothrix and Geobacter, these two groups of bacteria could exist in distinctive environmental niches defined by redox potential.
Project description:Redox potentials are a major contributor in controlling the electron transfer (ET) rates and thus regulating the ET processes in the bioenergetics. To maximize the efficiency of the ET process, one needs to master the art of tuning the redox potential, especially in metalloproteins, as they represent major classes of ET proteins. In this review, we first describe the importance of tuning the redox potential of ET centers and its role in regulating the ET in bioenergetic processes including photosynthesis and respiration. The main focus of this review is to summarize recent work in designing the ET centers, namely cupredoxins, cytochromes, and iron-sulfur proteins, and examples in design of protein networks involved these ET centers. We then discuss the factors that affect redox potentials of these ET centers including metal ion, the ligands to metal center and interactions beyond the primary ligand, especially non-covalent secondary coordination sphere interactions. We provide examples of strategies to fine-tune the redox potential using both natural and unnatural amino acids and native and nonnative cofactors. Several case studies are used to illustrate recent successes in this area. Outlooks for future endeavors are also provided. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.