Frontiers in metalloprotein crystallography and cryogenic electron microscopy

Metalloproteins comprise at least a third of all proteins that utilize redox properties of transition metals on their own or as parts of cofactors. The development of third generation storage ring sources and X-ray free-electron lasers with femtosecond pulses in the first decade of the 21st century has transformed metalloprotein crystallography. In the past decade, cryogenic-electron microscopy single-particle analysis, which does not require crystallization of biological samples has been extensively utilized, particularly for membrane-bound metalloprotein systems. Here, we explore recent frontiers in metalloprotein crystallography and cryogenic electron microscopy, organized for convenience under three metalloprotein-centered biological cycles, focusing on contributions from each technique, their synergy and the ability to preserve metals’ redox states when subjected to a particular probe.     

Probing metal-protein interactions using a de novo design approach

De novo design of metalloproteins provides a valuable tool for understanding the structural constraints and functional attributes of natural biological systems using first principles. This review focuses on recent research aimed primarily at probing the subtle interactions between metals and proteins in designed systems. Considerable attention has focussed on redefining novel design methods used in mimicking natural hemeproteins, mononuclear and dinuclear metallopeptides and functional biological electron-transfer proteins. The present results indicate that the field of metalloprotein design is contributing significantly to the understanding of metals in biology.     

Structural insights into protein-metal ion partnerships

New metalloprotein structures continue to provide discoveries regarding protein-metal ion partnerships. Many recent structures reveal metal ion sites that control or are controlled by protein conformational change, including modulation by alternative splice variants and striking conformational changes. Only a few novel catalytic metal centers have been revealed recently, such as the surprising Ni-hook superoxide dismutase catalytic site and the cubane-like Mn(3)CaO(4) photosynthetic oxygen-evolving center. However, important new variations on old heme themes, breakthroughs in the fields of metal ion regulation and metallochaperones, and captivating insights into partnerships between proteins and minerals have also been described. Very high resolution metal site structures and metalloprotein design will be increasingly important in order to leverage the wealth of native metalloprotein structures into a deep understanding of metal ion site specificity and activity.     

X-ray reduction correlates with soaking accessibility as judged from four non-crystallographically related diiron sites

X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.     

Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions.     

Protein engineering in bioelectrocatalysis

Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells and chemical syntheses. Escalating attention in this area is driven by remarkable progress in designing efficient interfaces for transferring electrons between electrode surfaces and redox proteins. Research in interface design is slowly shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering, which encompasses rational design, directed evolution and combined methods, offers many powerful methods and strategies for improving the electron transfer properties of redox proteins.     

Single-molecule fluorescence studies from a bioinorganic perspective

In recent years, single-molecule methods have enabled many innovative studies in the life sciences, which generated unprecedented insights into the workings of many macromolecular machineries. Single-molecule studies of bioinorganic systems have been limited, however, even though bioinorganic chemistry represents one of the frontiers in the life sciences. With the hope to stimulate more interest in applying existing and developing new single-molecule methods to address compelling bioinorganic problems, this review discusses a few single-molecule fluorescence approaches that have been or can be employed to study the functions and dynamics of metalloproteins. We focus on their principles, features and generality, possible further bioinorganic applications, and experimental challenges. The fluorescence quenching via energy transfer approach has been used to study the O₂-binding of hemocyanin, the redox states of azurin, and the folding dynamics of cytochrome c at the single-molecule level. Possible future applications of this approach to single-molecule studies of metalloenzyme catalysis and metalloprotein folding are discussed. The fluorescence quenching via electron transfer approach can probe the subtle conformational dynamics of proteins, and its possible application to probe metalloprotein structural dynamics is discussed. More examples are presented in using single-molecule fluorescence resonance energy transfer to probe metallochaperone protein interactions and metalloregulator-DNA interactions on a single-molecule basis.     

Redox biochemistry of mammalian metallothioneins

Metallothionein (MT) is a generic name for certain families of structurally rather variable metal-binding proteins. While purely chemical or biological approaches failed to establish a single physiologic function for MTs in any species, a combination of chemical and biological approaches and recent progress in defining the low but significant concentrations of cytosolic free zinc(II) ions have demonstrated that mammalian MTs function in cellular zinc metabolism in specific ways that differ from conventional knowledge about any other metalloprotein. Their thiolate coordination environments make MTs redox-active zinc proteins that exist in different molecular states depending on the availability of cellular zinc and the redox poise. The zinc affinities of MTs cover a range of physiologic zinc(II) ion concentrations and are modulated. Oxidative conditions make more zinc available, while reductive conditions make less zinc available. MTs move from the cytosol to cellular compartments, are secreted from cells, and are taken up by cells. They provide cellular zinc ions in a chemically available form and participate in cellular metal muffling: the combination of physiologic buffering in the steady state and the cellular redistribution and compartmentalization of transiently elevated zinc(II) ion concentrations in the pre-steady state. Cumulative evidence indicates that MTs primarily have a redox-dependent function in zinc metabolism, rather than a zinc-dependent function in redox metabolism.     

Manipulating redox systems: application to nanotechnology

Redox proteins and enzymes are attractive targets for nanobiotechnology. The theoretical framework of biological electron transfer is increasingly well-understood, and several properties make redox centres good systems for exploitation: many can be detected both electrochemically and optically; they can perform specific reactions; they are capable of self-assembly; and their dimensions are in the nanoscale. Great progress has been made with the two main approaches of protein engineering: rational design and combinatorial synthesis. Rational design has put our understanding of the structure-function relationship to the test, whereas combinatorial synthesis has generated new molecules of interest. This article provides selected examples of novel approaches where redox proteins are "wired up" in efficient electron-transfer chains, are "assembled" in artificial multidomain structures (molecular Lego), are "linked" to surfaces in nanodevices for biosensing and nanobiotechnological applications.     

Biogeographic and Evolutionary Patterns of Trace Element Utilization in Marine Microbial World

Trace elements are required by all organisms, which are key components of many enzymes catalyzing important biological reactions. Many trace element-dependent proteins have been characterized; however, little is known about their occurrence in microbial communities in diverse environments, especially the global marine ecosystem. Moreover, the relationships between trace element utilization and different types of environmental stressors are unclear. In this study, we used metagenomic data from the Global Ocean Sampling expedition project to identify the biogeographic distribution of genes encoding trace element-dependent proteins (for copper, molybdenum, cobalt, nickel, and selenium) in a variety of marine and non-marine aquatic samples. More than 56,000 metalloprotein and selenoprotein genes corresponding to nearly 100 families were predicted, becoming the largest dataset of marine metalloprotein and selenoprotein genes reported to date. In addition, samples with enriched or depleted metalloprotein/selenoprotein genes were identified, suggesting an active or inactive usage of these micronutrients in various sites. Further analysis of interactions among the elements showed significant correlations between some of them, especially those between nickel and selenium/copper. Finally, investigation of the relationships between environmental conditions and metalloprotein/selenoprotein families revealed that many environmental factors might contribute to the evolution of different metalloprotein and/or selenoprotein genes in the marine microbial world. Our data provide new insights into the utilization and biological roles of these trace elements in extant marine microbes, and might also be helpful for the understanding of how these organisms have adapted to their local environments.     

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.     

The dynamic thiol-disulphide redox proteome of the Arabidopsis thaliana chloroplast as revealed by differential electrophoretic mobility

The dynamics of the thiol–disulphide redox proteome is central to cell function and its regulation. Altered mobility of proteins in the oxidized and reduced state allows the MS-based identification of those thiol–disulphide proteins that undergo major conformational changes. A proteomic approach was taken with thylakoid-bound, luminal and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-less stromal subproteome fractions of the chloroplast from Arabidopsis thaliana . Among the 49 verified polypeptides were 22 novel redox proteins, previously not reported as being part of the redox proteome. Among the redox-affected proteins were PsbA (D1), PsaA1 and PsaF, chloroplast monodehydroascorbate reductase and also the Deg1 protease. Recombinant Deg1 and Deg2 revealed redox dependence of their proteolytic activity. The data provide new insights into the redox network of the chloroplast.     

Determination of in vivo disulfide-bonded proteins in Arabidopsis

Protein thiol-disulfide oxidoreduction plays an important role in redox regulation of cellular processes. Here we present a proteomic approach to visualize and map in vivo disulfide-bonded proteins in plants. A proteomic map of the disulfide-bonded proteins was achieved using 2D gel electrophoresis of Arabidopsis protein extract. Along with novel proteins identified as potentially redox regulated, we have also shown the feasibility of mapping some of the cysteines involved in the formation of disulfide bonds. This study presents an important tool for characterizing redox-regulated proteins.     

Insights into metalloproteins and metallodrugs from electron paramagnetic resonance spectroscopy

Metal ions play an important role in diverse biological processes, and much of the basic knowledge derived from studying native bioinorganic systems are applied in the synthesis of new molecules with the aim of diagnosing and treating diseases. At first glance, metalloproteins and metallodrugs are very different systems, but metal ion coordination, redox chemistry and substrate binding play essential roles in advancing both of these research fields. In this article, we discuss recent metalloprotein and metallodrug studies where electron paramagnetic resonance spectroscopy served as a major tool to gain a better understanding of metal-based structures and their function.     

Copper chaperones for cytochrome c oxidase and human disease

Biological processes in living cells are compartmentalized between lipid membranes. Integral membrane proteins often confer specific functions to these compartments and as such have a critical role in cellular metabolism and function. Cytochrome c oxidase is a macromolecular metalloprotein complex essential for the respiratory function of the cell. Elucidating the mechanisms of assembly of cytochrome c oxidase within the inner mitochondrial membrane represents a unique challenge for understanding metalloprotein biosynthesis. Elegant genetic experiments in yeast have defined several proteins required for copper delivery to cytochrome c oxidase. While the precise role of each of these proteins in copper incorporation remains unclear, recent studies have revealed that inherited mutations in two of these proteins can result in severe pathology in human infants in association with cytochrome c oxidase deficiency. Characterization of the molecular pathogenesis of these disorders offers new insights into the mechanisms of cellular copper metabolism and the role of these cytochrome c oxidase copper chaperones in human disease.     

Antioxidant enzymes as redox-based biomarkers: a brief review

The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease. [BMB Reports 2015; 48(4): 200-208]     

Thioredoxin - structural and functional complexity

Thioredoxins are small globular proteins that proved to be excellent model for investigating the relationship between the structure of protein and their physico-chemical and functional properties. The results from the experiments on thioredoxins offer the basic for the development of the new paradigms in the field of chemistry, biophysics and biology of proteins, with special attention to redox reaction in living cells, protein stability and design. It is a good example of broad class of sulphur-containing redox proteins.     

Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase

Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Differential scanning calorimetry of aposuperoxide dismutase and metalated superoxide dismutase unfolding transitions has provided quantitative estimates of the metal binding affinities for manganese superoxide dismutase. The binding constant for Mn(II) (K(Mn(II)) = 3.2 x 10(8) m(-1)) is surprisingly low in light of the essentially irreversible metal binding characteristic of this family of proteins and indicates that metal binding and release processes are dominated by kinetic, rather than thermodynamic, constraints. The kinetic stability of the metalloprotein complex can be traced to stabilization by elements of the protein that are independent of the presence or absence of the metal ion reflected in the thermally triggered metalation characteristic of these proteins. Binding constants for Mn(III), Fe(II), and Fe(III) complexes were estimated using quasireversible values for the unfolding enthalpy and DeltaC(p) for apo-Mn superoxide dismutase and the observed T(m) values for unfolding the metalated species in the absence of denaturants. For manganese and iron complexes, an oxidation state-dependent binding affinity reflects the protein perturbation of the metal redox potential.