Chapter 5 – Detailed Report on Bioinorganic Chemistry · • Spectroscopy of resting states, ......

Report from the Frontiers of Inorganic Chemistry Workshop 2002

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Chapter 5 – Detailed Report on Bioinorganic Chemistry Discussion Leader: Vickie DeRose Participants: Andy Borovik Brian Hoffman Dick Holm Steve Lippard Yi Lu Tom Meade Larry Que Hilary Godwin (in absentia) [J. Espenson, R. Eisenberg, Mahdi Abu- Ohmar, Tom Spiro were present for part of the second session.] I. BIOINORGANIC CHEMISTRY: SUMMARY Bioinorganic chemistry is a vibrant and dynamic field that heavily impacts developments in other fields. Its important successes include understanding metal-ion catalysis in enzymes, coordination chemistry of metals involved in imaging, diagnostics and therapeutics, and synthesis of small models. Current and future frontier research build on these successes and also on new discoveries of the past decade that have opened up entirely new areas. As advances in genetics, structural biology, spectroscopy and synthesis have bolstered recent efforts, new tools in bioinformatics, genomics, and nanotechnology will continue to advance the frontiers of the field. Bioinorganic chemistry is an excellent model of collaborative science that both contributes to and benefits from other fields. The educational component of this field is exceptional. II. BIOINORGANIC CHEMISTRY FOR THE COMING DECADE A. Recent Major Accomplishments

1. Structure and Function of Key Metalloenzymes

• New structures: cytochrome oxidase, nitrogenase, methane monooxygenase, hydrogenase, sulfite reductase

• Spectroscopy of resting states, intermediates • Gene expression systems

Figure 5.1 X-ray crystal structure of Methane Monooxygenase determined by Rosenzweig et al.


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The last ten years have seen extraordinary advances in obtaining resting-state structures of important metalloproteins using X-ray crystallography (Figure 5.1). These pictures of active sites are augmented by spectroscopic studies of trapped intermediates, and results from site-directed mutagenesis studies. The gene sequences for important enzymes have been cloned and sequenced, often with operons that include regulatory domains as well as the protein-coding sequences. This information has laid the groundwork critical to understanding the molecular mechanisms of a fundamental class of biocatalysts that perform transformations of potential economic value. 2. Biomimetic Chemistry

• Metal catalyzed hydrolysis • Models of the nitrogenase active site • Oxygen activation

Strong success has been achieved in synthesizing metal coordination compounds that mimic the structure and spectroscopic properties of important metal centers found in biological systems (Figure 5.2). These studies lead to an understanding of the requirements for forming catalytic sites in biology, and lead the way to creating new man-made catalysts. Great strides have been made in the past decade with respect to the development of structural and functional models of metalloenzymes that catalyze hydrolysis, nitrogen fixation, oxygen activation, and hydrocarbon oxidation. In the metallohydrolase area, the principles by which metal centers activate water to carry out amide and phosphate ester hydrolysis have been elucidated (J. Chin, S. J. Lippard). In the nitrogen fixation area, MoFeS clusters with stoichiometries approaching that of the FeMo cofactor in nitrogenase have been characterized (R. H. Holm, D.Coucouvanis) and some of these clusters can effect N-N bond cleavage of hydrazines (D. Coucouvanis). Furthermore N-N bond cleavage of dinitrogen has also been demonstrated for a tris(imido)molybdenum(III) complex (C. Cummins). In the oxygen activation area, there has been significant progress in understanding the chemistry of dicopper(I) centers. Crystal structures of two distinct dioxygen adducts have been obtained, one with µ-1,2-peroxo coordination (K. D. Karlin) and the other with µ-2,2-peroxo binding (N. Kitajima). Other dicopper(I) complexes react with O2 to cleave the O-O bond and afford high-valent CuIII

2(µ-O)2 complexes (W. B. Tolman, T. D. P. Stack, S. Itoh, M. Suzuki). In several of these cases, the O-O cleavage is reversible. Thus, not only can the O-O bond be activated and cleaved at dicopper centers, it can also be re-formed. Comparable progress has also been made in modeling oxygen activation at diiron centers. Crystal structures of three dioxygen adducts with µ-1,2-peroxo bridges have been reported (M. Suzuki, L. Que, S. J. Lippard). Also examples of Fe(III)Fe(IV) complexes derived from the reactions of diiron centers with O2 or H2O2 have also been found (L. Que, S. J. Lippard), providing models for high-valent intermediates for nonheme diiron enzymes. In one case, an Fe(III)Fe(IV) complex with an Fe2(µ-O)2

core has been crystallographically characterized (L.


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Que). At a recent international bioinorganic conference, the characterization of a diiron(IV) complex was presented (L. Que), which provides the synthetic precedent for the putative Fe2(µ-O)2

core of the diiron(IV) intermediate Q in the catalytic cycle of methane monooxygenase inspired by biological systems. 3. Chemistry of Bioinorganic Pharmaceuticals

• Anticancer therapeutics (Pt) • Contrast agents for imaging and diagnostics (Gd, Tc) • Metal pharmaceuticals as enzyme inhibitors

Fundamental studies of metal coordination properties have led to advances in the use of inorganic compounds as both therapeutic and and diagnostic agents.

Figure 5.2 Some successful bioinorganic models.


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3.1 Metal complexes as antitumor agents Cisplatin, cis-diamminedichloroplatinum(II) and carboplatin, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II), continue in widespread use as anticancer drugs. From detailed knowledge of how cisplatin binds its target in the cell, DNA, and how the resulting

adducts are processed (Figure 5.3) has come a rational design of new chemotherapeutic protocols that are now in a phase I clinical trial. Cisplatin analogues that can be administered orally and/or are active against cisplatin resistant tumors are now in the clinic. Multiplatinum complexes have been devised that work by a different mechanism and may be able to treat cancers for which cisplatin has not been very successful.

3.2 Metal complexes in imaging Coordination chemistry is providing entirely new generations of magnetic resonance (MR) and positron emission tomography (PET) contrast agents that report on anatomical and physiological processes (function) of intact animals and humans in the form of acquired 3D images. Metal complexes with favorable characteristics have been designed for MR imaging [Gd (T1), Fe (T2)] and PET (Tc, Cu). Specific advances include:

• cardiovascular imaging Gadolinium(III) bound to human serum albumin (HAS) allows this MR contrast ion

to reside in the blood long enough to provide high resolution images of the heart for diagnostic purposes.

• enzymatically activated MR contrast agents provide in vivo images of gene expression. Recent advances in the synthesis of new coordination complexes now allows the

synthesis of MR contrast agents that are enzymatically activated in vivo to conditionally enhance image intensity. When the access of Gd3+ to bulk water is limited, there is little effect on MR imaging but now agents designed to expose the Gd3+ by enzymatic processing in the body can be reliable markers for regions of enzyme activity. In these internally-activated contrast agents, covalently attached carbohydrate groups prevent Gd3+ exposure to water. When these gropus are lopped

Figure 5.3 Proposed mechanism for action of cisplatin.


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away from the macrocylic framework holding the Gd3+ by β-galactosidase. • MR imaging of secondary messengers in neuronal signaling

Ca2+ activated MR contrast agents detect in vivo Ca2+ concentration. Ca2+ is an important intracellular secondary messenger of signal transduction. Changes in the cytosolic concentration of Ca2+ trigger changes in cellular metabolism and are responsible for cell signaling and regulation. Advances in optical microscopy techniques and improvements in fluorescent dyes capable of measuring Ca2+ concentration have added greatly to the understanding of the critical role this ion plays in cellular and neuronal biology.

• PET imaging New contrast agents based on Tc and Cu coupled with rapidly developing

instrumentation is providing vivid images of both the stucture and function of organs in animals and humans. .

3.3 Bioinorganic Pharmaceuticals/Metal Complexes as Enzyme Inhibitors Transition metal complexes have long been known for therapeutic effects while the mechanisms of action are not understood. Advances in elucidating molecular structures of protein and enzymatic targets has led to a rational approach to the design and synthesis of new drugs. In addition to the Pt-anticancer drugs, many inorganic complexes are used in the therapeutic treatment of diseases, including: Hyperbilirubinemia (Sn, Zn), Nitric Oxide Transduction (enzyme inhibition) (Zn, Sn, Fe, Cu), Hypertension (enzyme inhibition) (Cu, Zn, Ge, Sb) Protease inhibitors (Co, Ti, Cu), Neurological effectors (Al, Fe, Zn, Pt, Sn), Arthritis (Au, Cu, Fe), Antiviral (enzyme inhibition) (W, Sn, V, Cu, Ni, Pd, Cu, Pd), and Hypochromic anemia (Fe). It must be emphasized that basic research of coordination complexes and enzyme active sites has led to much of the progress in these areas. 4. Long-range Electron Transfer

• Tunneling timescales (Ru-modified Cyt c) and pathways • Donor-acceptor partners

A fundamental understanding of long-range biological electron transfer is a triumph of the recent past. While the theory of these basic process had been formulated in the work of Marcus and others, and its quantum mechanical correlates, the connection between these theories and the actual process of electron transfer in biomolecules, both proteins and nucleic acids, presented a major challenge that has been addressed with great success. 5. De Novo Design in Metalloproteins and Peptides Successes have begun in two main areas of de novo design. Accomplishments in the last decade in the redesign of metalloprotein active sites are based on advances in protein expression methods, computer-based predictions of structural changes, and synchrotron access for protein crystallography. Based on these advances, tremendous progress has been made in this area. For


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example, several metal-binding sites, such as heme, zinc, binuclear iron centers and Fe-S clusters have been designed into de novo designed protein scaffolds such as four -helical bundles. Native protein scaffolds such as Greek Key -barrel have also been used for the design and engineering of zinc, copper, manganese, heme and non-heme iron (including Fe-S cluster) proteins. Furthermore, metal-binding sites have been also designed using peptides consisting of key structural elements for the formation of the sites such as Fe-S clusters and blue copper center. Complementary to these rational design approaches, new methods for selection and directed evolution of proteins in the laboratory have also been developed and resulted in metalloproteins with either improved function or new functions. As with the redesign of protein active sites, computer modeling is an integral component of this work. 6. Integration of Theory Coupling density functional theory calculations with experimental results has made a large impact on our understanding of the underlying principles of the extraordinary electronic and magnetic properties of metal centers in proteins. Molecular dynamics calculations have increased our understanding of the static and dynamic behavior of biopolymers including both proteins and DNA. Advances in the theoretical treatments of solvation have influenced and benefited {does this refer to DFT/MD or static/dynamic or proteins/DNA?]. 7. Influence of Bioinorganic Chemistry on Other Fields

• Zn-fingers and gene expression • Cu-SOD and disease • NO chemistry

Fundamental bioinorganic investigations have had broad impact in the areas of development, regulation, and disease. Examples include the ubiquitous Zn-finger motif, which is now known to have widespread importance in gene expression and regulation the connection between Cu/Zn superoxide dismutase and genetic mutations leading to Parkinson’s disease, and the discovery of nitric oxide as a central biological signaling agent with relationship to vasculature disease, the immune response and neuronal development. 8. New Discoveries That Lay the Basis for Future Research

• Role of metal homeostasis on disease • Biosynthesis of metal centers (i.e. urease) • Metals and prion disease • Metals and RNA (ribozymes)

From within the subdiscipline and from disciplines outside of chemistry, groundwork for the future hot topics in bioinorganic chemistry was laid in the last decade with the discoveries of biosynthetic pathways for metal centers, the influence of metals on complex nucleic acid structures and on RNA catalysis, hints at the complexity of biological metal sensing and


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transduction involved in controlling metal levels in vivo, and the most recent discovery that prion-type diseases may involve coordinated metal ions. The portfolio of bioinorganic research targets has increased over ten-fold with these discoveries from outside areas. B. At the Frontiers in Bioinorganic Chemistry 1. Biocatalysis

• Manipulation of small molecules N2, CO, O2, H2O, CH4, NO • Multi-e- reactions • Hydrolytic reactions • Relationship to green chemistries

An important and necessary development from the significant efforts in modeling how metal centers in biology work is the continuing design of metal complexes, either biomimetic or bio-inspired, that can catalyze transformations of interest to the chemical industry. Because of their biological roots, such catalysts have the potential of carrying out reactions under conditions that require low energy input and have significantly lower environmental impact, thereby giving rise to a generation of “green” catalysts. An example of a bio-inspired oxidation catalyst is a recently reported mononuclear chiral iron complex that reacts with H2O2 to catalyze the enantioselective cis-dihydroxylation of olefins (L. Que). Such a catalyst carries out the same transformation as the much more toxic OsO4 reagent and is inspired by the cis-dihydroxylation reactivity of arene degrading Rieske dioxygenases, which have mononuclear iron active sites. 2. Design and Synthesis of Functional Models

• New coordination chemistries • Unique functionalities (H-bonding, radicals) • Peptidomimetics • Engineering protein sites

Chemists aspire to understand natural phenomena by attempting to recreate them with as much fidelity as possible. In bioinorganic chemistry, the two broad categories of phenomena are active site structure and function, which usually is reactivity. In this way, the principles of structure stabilization and the factors essential to biological substrate reactivity may be revealed. Considerable progress has been made in e.g., iron-sulfur proteins and enzymes, molybdenum and tungsten enzymes, heme oxygenases and oxidases, and certain hydrolytic enzymes. Because of the increasing ability to interrogate protein-bound sites by spectroscopic methods with increasing information yield, targets of opportunity susceptible to the biomimetic approach must be chosen carefully. There remain, however, numerous problems to which this “minimalistic” approach can be applied with profit. These include, prominently, enzymes that catalyze multi-electron transformations such as all molybdoenzymes (excluding nitrogenase), hydrogenase, carbon monoxide dehydrogenase, and methane monooxygenase (2e-); Kodachrome oxidase (4e-); and nitrogenase, nitrite reductase, and sulfite reductase (6e-). It is to be emphasized that the chemist


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has not yet learned to manipulate substrates of these enzymes in the manner of enzymic reactions. Hence, biomimetic inorganic chemistry is a logical place to disclose the fundamental redox reactions of molecules such as H2, N2, CO, CH4, NO, and O2. [latter may be included in biocatalysis section] 3. Theoretical Analysis of Metalloenzyme Reaction Pathways As computational approaches become more efficient, mechanistic questions can be attacked by theoretical methods that include mixed quantum-mechanical and classical approaches. These, in combination with adequate solvation models, allow calculations of transition-state models that include hydrogen bonding and other effects not well modeled utilizing only the classical approach. Theoretical methods continue to be an important research tool. 4. Design and Synthesis of Functional Models The development of functional complexes that mimic chemical transformation found in nature continues to be an area of intense interest in bioinorganic chemistry. Examples encompass the activation of small molecules, such as dioxygen, carbon monoxide and dinitrogen. Two general approaches are used in designing synthetic metal complexes of this type: biomimetic and bioinspired. A biomimetic approach utilizes the exact structural elements found at the active site of metalloproteins to reproduce function. These efforts target complexes that emulate the primary coordination sphere of active site metal ions. A bioinspired approach uses some, but not all, of the structural properties of the active site to design new complexes with important functional properties. Incorporation of key structural elements into synthetic systems, particularly those in the secondary coordination sphere involving non-covalent interactions, is leading to a more complete understanding of structure/function relationships that are found in biomolecules. An example of this approach is the use of intramolecular hydrogen bonding to stabilized monomeric iron complexes with terminal oxo ligands derived from dioxygen (Borovik). Of the two broad phenomenological categories above, the creation of functional models carries the greater imperative. This work requires extensive use and possibly further extension of the principles of coordination chemistry. One example of new ligand types found in biology are stable radicals (attached to metals) that are directly implicated in the catalytic mechanism. There can be no doubt this general family of ligands and complexes will grow and new synthetic methods develop and further examples of radical -mediated enzymic reaction are discovered. A further area of assured significance in the future is “peptidomimetics”. Here peptides are designed with appropriate structural elements to stabilize natural metal-binding environments with physiological ligands. Further, residues can be varied, analogous to site-directed mutagenesis, so as to reveal their roles in structure and catalysis. Given the accelerating advances in peptide synthesis (including the concept of de novo design), protein structures, and protein dynamics, peptidomimetics is poised to play a prominent role in the next decade of biomimetic inorganic chemistry.


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5. Metal Site Biosynthesis

• Mechanisms of biological cluster assembly One of the major unsolved problems in metallobiochemistry is the elucidation of biosynthetic pathways leading to mononuclear and polynuclear metal sites. This is truly an interdisciplinary problem that will require contributions from geneticists, biochemists, biophysicists, and inorganic chemists. Almost nothing is known about the mechanism of metal ion insertion and the means of cluster formation in proteins. Substantial progress is being made on several fronts, e.g., the binuclear site of urease and the cofactor clusters of nitrogenase. Yet much remains. The role of the inorganic chemist surely will be to discover new reactions leading to biological coordination units and to demonstrate new structures relevant to protein-bound metal sites. The problem of metal site biosynthesis promulgates a forefront area in inorganic synthesis. 6. Metal Trafficking

• Chaperones and insertases • Disease states

Understanding how metals are regulated and trafficked in cells requires the marriage of bioinorganic coordination chemistries, in-situ spectroscopies, X-ray crystallography, and molecular and cellular biology. The challenges of tracing metal ion pathways towards their fates in metalloenzymes (see above) or disposal as toxic agents will continue to require the cutting edge of all areas of chemistry, and connects inorganic chemistry with the overarching area of ‘biocomplexity.’ 7. Electron Transfer The study of biological electron transfer (ET) has tended in two parallel directions, with one being the examination of ET between two redox centers within a single macromolecule, often with a second center having been introduced synthetically, the other being the study of ET between donor-acceptor partners. The future will bring major efforts in both directions. In the first, a key focus likely will be on the role of ET between naturally occurring redox centers in proteins/enzymes that contain multiple centers as a conduit to carry electrons between the site of electron entry/exit on the protein surface and the enzymic active site. The second will include the characterization of the interactions between strongly-interacting partners, but will accentuate the dynamics of docking and recognition of more weakly interacting ones. A third and emerging theme will be to design ways to utilize long-range ET to photoinitiate enzymatic redox reactions and to study their reaction intermediates. 8. Metals in Medicine The primary barrier to the exploitation of new imaging modalities for basic biological research and in clinical settings remains the design and synthesis of new coordination complexes as


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contrast agents. MR, PET, and traditional radiopharmacutical imaging modalities are undergoing dramatic changes by the advance of new technologies. Modern optical microscopy techniques, combined with fluorescent indicator compounds, have revolutionized studies of biological structure, function and development by allowing the physiology of intact cells and tissues to be assayed. However, light-based techniques, ranging from video-microscopy to laser scanning confocal microscopy, work best in the outermost 100 µm of a biological tissue due to light scattering and uncorrected optical aberrations.

• MRI of biological structures offers an alternative to light microscopy that can circumvent these limitations and analyses demonstrate the feasibility of true three-dimensional MR imaging at cellular resolution (~ 10 µm). In order to exploit the power of MRI for biological (and ultimately clinical studies), new contrast agents must be designed and tested that report on structure and function (i.e. gene expression). See Figure 5.4.

Figure 5.4 Schematic representation of an enzyme-activated MR contrast agent. A. diagram representing the site-specific placement of the galactopyranosyl ring on the tetraazamacrocycle (side view). Upon cleavage of the sugar residue by b-galactosidase (at red bond), an inner sphere coordination site of the Gd3+ ion becomes more accessible to water. B. Space-filling molecular model (top view, from above the sugar residue) of the complex before (right) and after cleavage by the β-gal (left), illustrating the increased accessibility of the Gd3+ ion (magenta) upon cleavage.


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The challenge for the development of new agents is a fundamental structural analysis of how to modify the chelate structures to accommodate targeting groups such as peptides and DNA and to improve image contrast and delivery. 9. Metalloenzymes with New Activities

• Directed evolution • Active site engineering

By a recent estimate, metalloproteins account for approximately half of all proteins in biological systems. They play key roles in all almost every aspect of biology. The design and engineering of novel metalloproteins with desired structural and functional properties remains one of the most exciting research areas. Protein design is not only an ultimate test of our knowledge of metalloproteins, but also can result in new metalloproteins with improved or even unprecedented properties for biotechnological and pharmaceutical applications that benefit the society. In the next decade, the development of new computer algorithms for rational design, and innovative directed evolution/selection strategies will assist protein design. We will also see more studies that combine both rational and evolution approaches. 10. Metals and Nucleic Acids

• Coordination modes • Ribozyme mechanisms

The importance of nucleic acids in a myriad of cell functions coupled to the relative youth of the field of nucleic acid structural biology make this a forefront field in bioinorganic chemistry. It is clear that metal ions are intimately involved with the structures and chemical functions of nucleic acids, including both RNA and DNA. Recent crystal structures of complex RNA molecules, from ribozymes to ribosomes, show localized metals whose influence on solution-state structure and function has yet to be determined. Spectroscopic and synthetic endeavors in measuring and modeling metal-nucleic interactions are current ‘hot topics’ emphasized in every national and international meeting. Based on groundwork currently being obtained, future work is expected to include design of metal sites that may stabilize unique structures, including supramolecular lattices, and also create new catalytic functions. 11. Neurochemistry – Current Topics and Future Challenges Metalloneurochemistry: An Emerging Area of Bioinorganic Chemistry

“…Of the areas at the interface between inorganic chemistry and biology that remain to be explored, the role of metal ions in neuroscience is perhaps the most prominent. Bioinorganic chemists have been attracted historically to systems involving transition metal ions because of their valuable magnetic and spectroscopic properties. Despite the preponderance of Na+, K+, Mg2+ and Ca2+ in biological processes, the inorganic


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chemistry community has often ignored these metal ions because they lack the electronic and magnetic properties of their d-block counterparts. In a similar manner, neuroscientists have focused on group 1 and 2 metal ions and, until recently, have dismissed transition metals such as Mn, Fe, Cu, and Zn as inconsequential trace elements in the central nervous system [see Figure 5.5]. By combining the ability of bioinorganic chemists to evaluate the properties of metal ions with that of neuroscientists to explore the physiology of the nervous system, a powerful new alliance could emerge for understanding such complex processes as neurotransmission and synaptic plasticity. An important consequence would be to uncover the causes of, and develop treatments for, neurodegenerative disease….” From: Coordination Chemistry for the Neurosciences. Shawn C. Burdette and Stephen J. Lippard, Coord. Chem. Rev., 216-217, 333-361 (2001).

Figure 5.5 Hippocampal brain slice from rat stained with Zinpyr-1 showing vesicular zinc in CA3 nerve terminals; photo courtesy of Dr. Chris Frederickson, NeuroBioTex.

Specific topics that are currently being explored are: • the role of zinc in vesicles housed in the presynaptic nerve terminals of specific cells in

the hippocampus, the center of learning and memory in the brain; • the coordination chemistry that leads to the selective passage of ions through channels

and pumps • the role of nitric oxide as a retrograde messenger in the postsynaptic terminals of the

brain; • the role(s) of metal ions in diseases that arise from mutated or misfolded proteins,

including Alzheimer’s, prion disease, and ALS.

To meet these new challenges, some novel tools have to be developed for studying specific problems. For example, there is currently no good way to monitor where NO is generated and


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how far it migrates in brain tissue. Soluble sensors for in vivo use would greatly facilitate investigations of this problem. These sensors could be designed to take advantage of the coordinating ability of NO to transition metal complexes. Collaborations between inorganic chemists and neuroscientists are essential for significant progress to be made in the field of metalloneurochemistry. It is likely that, given the interest of young scientists in the brain and nervous systems, this topic will be of increasing significance in the coming decade. 12. Biological NO chemistry The medically critical production, regulation, and mode of action of nitric oxide as a signalling agent is an area of bioinorganic chemistry that encompasses structural biology, catalysis, and small-molecular chemistry. Bioinorganic mechanistic work on the key enzymes for NO production (NO synthase) and its target messenger agent, guanylate cyclase, remains a critical and provocative field that has relied heavily on past bioinorganic success stories in the field of heme enzymes. Laboratory investigations of the stability and reactivity of NO(x) compounds such as peroxynitrite, the toxic product used in cell defense systems, are also ongoing challenges that bridge inorganic and medicinal chemistries. 13. Sensors

• Coordination models for detecting metals in, ex vivo • Zn and Ca sensors for imaging metals in development, neurochemistry, disease states • Biomolecules as metal sensors • Bioinorganic systems as biosensors (ex. redox-based sensors)

Sensors for metal ions are an important focus of current research. These include chemosensors and biosensors. Synthetic calcium sensors have enjoyed the most success in monitoring in vivo calcium distributions during the cell growth and metabolism. Other chemosensors for zinc and mercury have also been synthesized and studied. Proteins, peptides, catalytic DNA/RNA have also been developed as biosensors for zinc, lead and copper. Culture cells that can sense different heavy metal ions have also be used. Metal ion sensors will be one of the most active areas of research in the next ten years, as they will be applied to environmental monitoring, clinical toxicology, neurological science, and wastes management. DNA oligonucleotides are a revolutionary addition to the sensor field. The study of energy and electron transfer processes through DNA duplexes and the development of DNA hybridization probes and electrochemical sensors have resulted in the incorporation of numerous transition-metal complexes into DNA oliognucleotides. These include ruthenium, osmium, iron, rhodium, and copper complexes.


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Ferrocene (Fc) and its derivatives are attractive electrochemical probes because of their stability and convenient synthetic chemistry. Fc-containing DNA oligonucleotides have been prepared by attaching ferrocenyl moieties to the 5’ termini through either solid phase synthesis using phosphoramidites or by reacting suitable ferrocenyl derivatives with end-functionalized oligonucleotides. Efforts have focused on ways to develop microsensors for electronically detecting nucleic acids where ferrocenyl derivatives are site-specifically incorporated into DNA oligonucleotides and are used as probes (Figure 5.6). . 14. Biomaterials

• Biomineralization • Biomimetic materials • Adhesion

Important hot areas of current research in biomaterials include:

• understanding the inorganic chemistry of adhesion in mollusks (Wilker) • using biomolecules, such as ferritin, as templates to synthesize new materials

(Douglas) • understanding the properties of shell growth in mollusks (Stuckey) • using inorganic materials in combination with biomolecules for the development of

new sensors (Mirkin) • using combinatorial methods to discover new metallobiomaterials (Belcher).

C. New Frontiers in Bioinorganic Chemistry 1. Inorganic chemistry of the Cell (‘Metallome’) Similar to the proteome and genome, determining the actual inorganic composition of cells under different expression conditions will be required to truly understand the influence of metals and other inorganic constituents in biological systems. New analytical methods and the fundamental methodology derived from nanotechnology will strongly impact determination of the ‘metallome’.

Figure 5.6 Electrode-based Biosensors based on ferrocene-linked DNA arrays


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2. Incorporating genomics into bioinorganic chemistry

• New metalloproteins with novel functions discovered from gene sequences • Chip detection of gene expression • Expression in response to (toxic) metals (Pb, Cd, Hg, Cr) • Metalloprotein expression related to development, disease, cell cycles • Metalloproteins involved in signaling • Metal ion homeostasis • New levels of biocomplexity

The Likely Impact of Genomics on Bioinorganic Chemistry The advent of gene chips, by which one can monitor gene expression, is likely to have a major impact on the field of bioinorganic chemistry. Because the area is so new, it is difficult to imagine exactly how this process will unfold, but a few examples will be useful to illustrate the concept. In the area of metals in medicine, it will become possible to compare the relative expression of specific genes in diseased and normal tissue from the same source, in the presence and absence of a particular metallopharmaceutical. From such information might come clues about the mechanism of action of the drug or new strategies for treatment. Another example pertains to the investigation of a cellular process such as apoptosis, or programmed cell death, which involves many metalloenzymes housed in the mitochondrion. The concerted expression or suppression of genes that encode these enzymes could allow one to understand the underlying bioinorganic chemistry of the process. A different application would be in the area of metalloregulation of gene expression, where genomics would enable new systems to be understood in response to a toxic metal ion such as lead in the environment. Finally, it should be noted that inorganic chemistry might play a role in the field of genomics by providing modified surfaces and new materials that could be used for the development of smarter, more efficient gene chips. 3. Biomimetic chemistry

• linked chemistry (sequential reactions, triggering) • scaffolds • combinatorial approaches • confined spaces • mesoporous solids, vesicles, nanotubes

Both biomimetic and bioinspired approaches lead to complexes that serve as the springboard for discovering more efficient reactions that are useful to chemists. Future challenges will have to incorporate the more structural elements found in both the primary and secondary sphere found in metal proteins. This includes using non-covalent interactions to influence chemical reactivity. These interactions are essential in modulating chemical reactions, especially for selective reactivity. Methods to accomplish this are still needed. One promising area is in the de novo


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design of metallpeptides. DeGrado and Dutton's work on electron transfer in de novo designed metallopeptides illustrates the utility of this approach. However, functional systems for chemical transformations are still lacking. Future efforts will be on developing methods to make new ligands, scaffolds or peptides that yield a diverse group of metal complexes. While rationally designed systems are still essential in future discovery of this type, combinatorial methods cannot be overlooked. Rapid methods for making and screening a large array of complexes are needed to advance this area. A second approach that will be important in the future is developing bioinspired complexes in confined spaces. The goals are similar to those above, i.e, to making synthetic systems that reproduce or enhance the chemistry found in nature. This area encompasses a wide variety of synthetic disciplines, but, in the near future, most likely will combine synthetic inorganic chemistry with material science. The merging of efforts in these areas (breaking down unnecessary barriers between disciplines) is necessary for a productive future. 4. Environmental bioinorganic chemistry

• Oceans • Extreme environments (vents) • Toxic metals (Se, As)

The involvement of bioinorganic complexes in complex environmental systems will be an important, and wide-open, area of future research. 5. Dynamics of metalloproteins

• Motions related to catalysis • Docking

While most current structural biology is based on ground-state structures, the dynamics of biological systems and relationship of motions to function remain a forefront area of research. III. FINDING THE RIGHT BALANCE A. What is the right balance between fundamental and directed research; multi-

investigator vs. single-investigator grants? 1) We are already inspired by fundamental problems in Biology and by societal needs in

medicine, energy, and the environment.

2) This approach has been successful, in that discoveries from fundamental research have already proven critical to other scientific disciplines and to technology breakthroughs.


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Examples: - • Zn and development • Sensors based on electron transfer

B. What is the proper balance between single- and multi-investigator approaches? This discipline of bioinorganic chemistry is already highly collaborative

• Predominant mechanism is through single-investigator projects with arranged collaborations

• Mechanism is already very successful The group favors primary funding for single-investigator driven research

Autonomy: • drives timely progress • allows for flexibility in collaborations required for fast response to new discoveries

Circumstances appropriate for formal multicollaborative grants include:

• Defined, established questions • Need for motivation and resources for highly integrated and synchronous research • Widely disparate fields and long-term questions

Example areas: -

• inorganic chemistry in neuroscience • biogeochemistry

C. Can the hot areas of the future (as identified above) be worked into existing NSF

initiatives?

Not really addressed; all members of group have single-investigator ‘basic research’ funding from NSF D. Can the identified hot areas be used to develop other initiatives across NSF (not just

within IBO and CHE, but across other divisions as well)? Potential example areas:

• inorganic chemistry in neuroscience • biogeochemistry

E. How can the inorganic community work together most effectively to take advantage of

existing initiatives and promote new ones?


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Not addressed

F. Balance' Summary The field of bioinorganic chemistry has thrived precisely because it is a highly interactive, collaborative group that is driven by research needs to constantly push for new techniques and partnerships. This success has been met, however, in the context of investigator-initiated and single-investigator grant programs that are based on problems in basic research. Single-investigator grants require, and therefore produce, high-impact results produced in a timely fashion. This structure also allows flexibility to collaborate as needed. Potential areas were identified in which structured collaborations could be beneficial. IV. FURTHER REMARKS: A. Opportunities for inter-group scientific interactions

• Biocatalysis related to design of new catalysts, industrial processes • Environmental chemistry:

o Bioremediation o Microbial-mineral interactions o Biogeochemistry o Toxic metals

New frontiers such as inorganic chemistry neuroscience and inorganic biogeochemistry have a strong chance of thriving if this field is ensured continued support for basic research. B. A New Theme/Title for the Area: Inorganic Chemistry of Life Examples for public dissemination

• Lance Armstrong and Pt compound • In vivo image and Gd/Tc compound • Crops and nitrogenase MoFe cofactor • Prions and Cu • Nerve cell and K+ channel (+ detail of K+ ligands) • Valdez bioremediation • Egg/Ca2+ image for biosensors • Toxic metals- paint and children • Antibiotics: Tb-KatG story • Fe, Klausner

C. Bioinorganic Chemistry and Education Bioinorganic chemistry is one of the most effective disciplines for training students in


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fundamental topics and the broader scope of science. Students in this area are trained as scientists experienced in the power of collaboration for achieving goals. Students with small-molecule modeling projects in particular receive desirable training in synthesis and evaluation, as well as education in biological systems. This field has a strong tradition of mentoring through the Graduate Student Gordon Research Conference and previously NSF-supported Summer Workshop in Bioinorganic Chemistry (Georgia). D. Existing and Emerging Techniques Critical For and Stimulated By the Field

• Higher sensitivity in diffraction- detectors, sources • High-resolution (sub-angstrom) protein crystallography • Cryocrystallography • Crystallography of trapped intermediates • NMR at high fields • ENDOR (and ESEEM) Spectroscopies • Low temperature trapping of chemical reaction intermediates (modeling chemistry,

Davydov/Hoffman cryoreduction of proteins) • VF/VT MCD • Mossbauer • Magnetism and theory • X-ray absorption spectroscopy • Rapid kinetics • Single-molecule folding and reactions • Mass spectroscopy • reaction products, models • posttranslational modification • protein-protein and protein-DNA interactions • folding and accessibility

Chapter 5 – Detailed Report on Bioinorganic Chemistry · • Spectroscopy of resting states, ......

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