Applications of Physical Methods to Inorganic and Bioinorganic Chemistry

By Robert A. Scott

Modern spectroscopic and instrumental techniques are essential to the practice of inorganic and bioinorganic chemistry. This first volume in the new Wiley Encyclopedia of Inorganic Chemistry Methods and Applications Series provides a consistent and comprehensive description of the practical applicability of a large number of techniques to modern problems in inorganic and bioinorganic chemistry. The outcome is a text that provides invaluable guidance and advice for inorganic and bioinorganic chemists to select appropriate techniques, whilst acting as a source to the understanding of these methods.

This volume is also available as part of Encyclopedia of Inorganic Chemistry, 5 Volume Set.

This set combines all volumes published as EIC Books from 2007 to 2010, representing areas of key developments in the field of inorganic chemistry published in the Encyclopedia of Inorganic Chemistry. Find out more.

 

• Language: English
• Publisher: Wiley-Interscience
• Release date: Feb 19, 2013
• ISBN: 9781118625286

Book preview

Series Preface

The success of the Encyclopedia of Inorganic Chemistry (EIC) has been very gratifying to the editors. We feel, however, that not everyone will necessarily need access to all ten volumes of the Encyclopedia. Some readers may prefer to have more concise thematic volumes, targeted at their specific area of interest. This idea has encouraged us to produce a series of EIC Books, focusing on topics of current interest. These will appear on a regular basis from now on, and will feature the leading scholars in their fields. Like the Encyclopedia, we hope that EIC Books will give both the starting research student and the confirmed research worker a critical distillation of the leading concepts, and provide a structured entry into the fields covered.

Computer literature searches have become so easy that one could be led into thinking that the problem of efficient access to chemical knowledge is now solved. In fact, these searches often produce such a vast mass of material that the reader is overwhelmed. As Henry Kissinger has remarked, the end result is often a shrinking of one’s perspective. In the EIC Books we hope readers will find an expanding perspective to furnish ideas for research, and a solid, up-to-date digest of current knowledge to provide a basis for instructors and lecturers.

I take this opportunity of thanking R. Bruce King, who pioneered the Encyclopedia of Inorganic Chemistry, my fellow editors, as well as the Wiley personnel and, most particularly, the authors of the articles for the tremendous effort required to produce such a series on time. I hope that EIC Books will allow readers to benefit in a more timely way from the insight of the authors and thus contribute to the advance of the field as a whole.

Robert H. Crabtree

Yale University

Department of Chemistry

April 2007

Volume Preface

All areas of inorganic chemistry depend on a variety of physical methods and instruments to characterize molecules and materials, and their reactions. It is difficult for newcomers to the field, and even experts in allied fields, to recognize the utility of a given physical method to provide useful characterization of their particular system. This is true regardless of whether one is synthesizing inorganic or organometallic molecules, studying solid-state materials, investigating surface species, or purifying and characterizing a metallobiomolecule. Characterization of molecular structure and reactivity in any of these cases usually requires the application of several complementary physical methods, often provided by different collaborators, each of which provides an essential piece of the puzzle. Researchers need a mechanism by which they can both quickly identify which physical methods would be useful and contribute to an intelligent interpretation of the results.

This book is intended to fulfil this purpose. It provides a practical introduction to a comprehensive set of physical methods that have been applied to inorganic systems, with an emphasis on answering questions like, ‘‘what kind of information would this method provide?’’ or ‘‘what constraints are there on the type of sample that can be examined?’’ and, more importantly, ‘‘what information will not be available if I employ this method (i.e., what are its limitations)?’’ This book provides a relatively quick reference guide (like the ‘‘Quick Start’’ manuals that come with computers and peripherals these days) that nonexperts can consult to select a set of physical methods appropriate for the characterization of a given material. Although other sources describing the theory and application of several related methods are available, this book provides the investigator with a more comprehensive and uniquely practical resource for physical method applications.

Another unique feature of this book is its association with the Encyclopedia of Inorganic Chemistry (EIC), which has been published in first and second Editions in print, and most recently in online format. We believe that this book is a unique and powerful resource by itself, but all the articles within it will also become components of the ‘‘living’’ online Encyclopedia, which provides a format for continual updating and augmentation. The editors would appreciate receiving suggestions from the inorganic chemistry community about additional methods worthy of inclusion in the online Encyclopedia. Only with such continual expansion will this resource maintain its comprehensive nature.

We believe that the practical nature of these descriptions of physical methods makes this book uniquely accessible and useful. Each physical method is introduced by a Method Summary. These summaries are structured consistently to answer very practical questions about what information is and is not available from each method and what kinds of materials can be probed. Thus, at a glance, a nonexpert can identify a subset of potentially useful methods, and then concentrate on those articles to determine which methods will be most useful in solving their problem and to see specific examples of how that method is utilized. Researchers will obtain sufficient information to determine if collaboration with others is the best course of action. Our goal is not to create experts in any given method. We have requested that our authors not include overwhelming theoretical detail. Should a researcher find a particularly useful and interesting method to employ, there are other resources that can be used for a deeper understanding, and the articles provide a list of annotated reference material and detailed bibliographies for further study.

R.A.S. acknowledges that the concept of the Method Summary was born in a set of Inorganic Biochemistry Summer Workshops offered in the 1990s in Athens, Georgia, by the Center for Metalloenzyme Studies. These 10-day workshops invited expert trainers to work with 50–60 student participants on a given technique (most of them biophysical) and R.A.S. designed these one-page practical summaries as a requirement for the introduction of each lecture. We thank the trainers in those workshops (some of whom are authors in this book) for feedback on the design of this summary.

We very much hope that you find this compilation useful and encourage your feedback and suggestions of additional methods that could be included.

May 2007

Circular Dichroism (CD) Spectroscopy

P. Anthony Presta and Martin J. Stillman

University of Western Ontario, London, ON, Canada

METHOD SUMMARY

Acronyms, Synonyms

Circular Dichroism (CD)

Optical Rotatory Dispersion (ORD).

Measured physical quantities

CD spectroscopy measures the difference in absorbance between left- and right-circularly polarized incident radiation, in the near ultraviolet to visible and infrared regions of the spectrum.

ORD uses plane polarized light and provides the degree of rotation of plane polarized light as an angle.

Information available

When ORD can specifically provide information it is mentioned, otherwise the following information is for CD primarily:

presence of chiral chromophores (both CD and ORD)

relative amounts of α-helix and β-sheet secondary structure in protein molecules

changes in all degrees of structure in biological molecules

denaturation as a function of structure, readily determined using CD spectral data as a function of a physical (e.g., temperature) or chemical (e.g., pH, denaturant) change

monitoring of structural and metal ion-ligand changes in metalloprotein complexes by titration experiments

metal-ligand stoichiometry

metal-ligand coordination geometry.

Information not available, limitations

Chromophore must be chiral for any information to be obtained.

Metal-ligand binding information is inferred from changes in spectral titrations, but specific bonds between a metal atom and its ligating atoms cannot be assigned.

Metal atoms with high energy absorbance bands (that absorb in the far ultraviolet) cannot be directly probed; these include the alkali metals and alkaline earth metals.

Examples of questions that can be answered

What are the stoichiometries of the major complexes between a metal ion Mn+ and a protein ligand?

How does metal substitution occur when a different metal ion with higher binding affinity to the protein is added to a solution of an existing metal-protein ligand complex?

How does the protein morphology or secondary structure change upon metal binding?

What are the identities of the protein atoms that bind to the metal ion (e.g., Cotton effect question, especially for Fe binding)?

Is the compound analytically pure?

Is the chirality opposite that of the enantiomer?

Major advantages

The method obtains highly reproducible information on metal-ligand changes as the titration experiment proceeds.

CD signals can be compared to the analogous absorbance spectra for distinguishing metal binding.

Chiral chromophores are discernible in samples with multiple overlapping absorbance bands.

Sample can be combined with other spectroscopic techniques in ‘‘real time’’.

CD spectra in particular are immune to solvent effects and therefore can provide exceptionally good data on the chirality of a molecule with absorption bands distributed throughout the optical spectrum.

Major disadvantages

Much of the structural information for metal-ligand binding (coordination number, stoichiometry) is inferred by the titration; confirmation of structural information is required by other techniques.

Analytical uses cannot be estimated prior to use of a pure sample. Estimation of the rotation (in ORD) and the signal intensity (in CD) cannot be carried out.

The absolute sign of the rotation or the CD signal cannot be calculated (+ or −) currently.

Sample constraints

Soluble samples are the easiest.

Scatter from frozen samples can cause significant baseline deviations in CD experiments.

Samples must contain a chiral chromophore that absorbs in the near ultraviolet to infrared region.

1 INTRODUCTION

Circular dichroism (CD) spectroscopy is a method that measures the difference in the absorbance of left- and right-circularly polarized light as a function of the energy of the radiation (but usually reported as the wavelength of the light) by chiral chromophores. The absorbing chromophore may be a chiral center due to asymmetric coordination chemistry, as in the example of the chiral Cotton effect due to the asymmetric coordination chemistry of iron complexes. Alternatively, the chromophore may display an induced chirality brought about by the formation of three-dimensional bonding structures. This is the case in chiral structures such as the α-helix or β-sheet conformations found in peptide chains, and commonly referred to as the secondary structures of proteins and polypeptides. Similar delocalized chiral chromophores resulting from induction of the specific three-dimensional structure are also observed in metal-ligand clusters. These structures are commonly found in proteins chelating a variety of metal ions. Moreover, the variety of possible metal-binding side-chain functionalities in the amino acids within the protein chain, the changes in metal-ligand stoichiometry, variable metal coordination chemistry, and the possibility of intermolecular cross-linking all can result in changes to the three-dimensional structure of the chromophore, with concomitant profound changes in the resultant CD spectrum of the sample. These specific changes in the CD spectrum allow detailed study of metal-binding properties.

There is a formidable and varied array of metal ions that participate in biological systems, both as essential cofactors within the cells and organs of the living organism or as toxic elements deleterious to the organism. Metal ions in vivo are principally found chelated to proteins and polypeptides, owing to the wide variety of negative dipole functional groups (usually with oxygen, nitrogen, or sulfur as the ligating atom from the amino acid side chains of the protein) attracted to the positive metal ions. A great deal of research has been conducted and remains ongoing to define and elucidate the roles and unique chemistry that the various metal ions play in biological systems, specifically as metal-protein complexes.

In general, the type of information that is of greatest interest when discussing metal-protein chemistry include the following: (1) changes in stability of the protein resulting from changes in protein secondary structure; (2) identification of the ligating functional group of the protein; (3) the oxidation state of the metal ion; (4) the coordination number of the metal ion; (5) the metal-to-ligand stoichiometry; and (6) changes in metal-protein reactivity.

2 TECHNICAL BACKGROUND

2.1 Measuring CD Spectra

The easiest way to obtain a CD spectrum is to use a spectropolarimeter. In these instruments the signal is plotted as the ellipticity of the emerging radiation after passage through a sample, θ = (2.303/4)(Al − Ar) with units of radians as a function of the wavelength of the light, θ vs. λ in units of nm usually.

When reduced to units of degrees, θ = 2.303(Al − Ar)(180/4π) = 32.98ΔAL−R

The molar quantity is given by: [θ] = 3298(εL − εR) = 3298ΔεL−R, where the 3298 converts the L M−1 cm−1 unit of molar absorptivity (ε) to the 0.1 L M−1 cm−1 units of molar ellipticity.

It is easier, and to some extent more useful, to quote CD spectral data in terms of ΔAL−R for solutions without known concentrations and ΔεL−R when concentrations are known. This then can match data from the associated magnetic CD spectroscopy (see Magnetic Circular Dichroism (MCD) Spectroscopy).

Reporting CD spectral data as ΔεL−R vs. λ in nm is good practice.

Most CD spectropolarimeters must be calibrated. A common way is to compare the absorption and CD spectra of ammonium (+)-d10-camphorsulfonate (ACS) using a solution nominally 1 mg mL−1 in a 1 mm cell. Using the absorption at 285 nm (ε285 nm = 34.5M−1 cm−1) to determine the concentration, the positive CD band at 290 nm may be used for calibration Δε291 nm = 2.36. A negative band at 190 nm can be used as well to extend the calibration into the UV region.

Of major concern in measuring CD spectra, is the absorbance of the solvent. Whereas in the electronic absorption spectrum (see Electronic Spectroscopy) all components can be measured if the blank is simply set to be the empty cuvette or air in the double beam instrument, in the CD experiment, the nonchiral components are not ‘‘seen’’ or recorded in the CD spectrum yet they absorb light and can easily reduce the CD spectral data to useless lines. In reality, this only becomes a major problem when solvents and buffers begin to absorb below about 300 nm. Although, we should note that great care should be taken to keep the absorption low (may be less than 0.6) for the CD spectra of porphyrinoids, especially in the Q–band region from 580 to 800 nm. However, the problem is insidious in that after the measurement has been made, it is not possible to determine if the background absorbance precluded accurate measurement.

What goes wrong? The CD intensities start being nonlinear with respect to concentration of the absorbing species when the background absorbance (and instrument insensitivity) results in the controlled DC voltage of the photomultiplier tube losing its lock. Most instruments maintain this DC voltage by changing the applied dynode volts of the photomultiplier tube within its working range. The gain on the photomultiplier tube is increased by increasing the dynode voltage, which concomitantly increases the noise of the measurement. As the light striking the photomultiplier tube decreases (because of increased background absorbance), so the dynode voltage increases. When no more gain can be obtained, the signal starts being nonlinear and eventually the photomultiplier reports totally erroneous CD signals. Remembering that an absorbance of 1 allows transmission of only 10% of the incident light, and an absorbance of 2 allows transmittance of just 1% of the incident light, it is easy to see that with solvent or buffer, most of the light may be absorbed. CD spectrometers typically do not measure linearly for total sample absorbance above 1.0.

2.2 Choice of Cuvettes

Continuing with the theme of obtaining CD spectral data unaffected by the nonchiral components, including the solvent, leads us to consider the cuvette. As described above, one must be very careful to check that the cuvette, solvent, and all other components do not absorb significantly in the region of interest. This becomes a concern in the UV- and IR-regions of the spectrum. As an example, for spectra below 190 nm, fused silica windows are beneficial and the sample compartment must be purged of O2 with N2 gas, because O2 has absorption lines near 178 nm (O2 must also be purged from the lamp and monochromator to reduce the build up of O3). Because the concentration of the solvent (including all components, notably buffers) is so high, cells with pathlengths down to 0.01 cm should be used. For all measurements below 200 nm, it would be wise to use a series of cuvettes, with pathlengths of 0.05, 0.1 and 0.2 cm to confirm linearity of the CD signal.

Strain in the cuvette (from the windows themselves, from misalignment, etc.) can result in significant distortions in the CD spectrum recorded. It is essential that a solvent blank be measured with the cuvette in exactly the same orientation to be used with the sample. It is good practice to mark the cuvette so it is placed with the same window facing the lamp. Many other factors can contribute to depolarizing distortion that will result in incorrect spectral data; it is wise to be very careful with cuvette choice and testing, especially below 200 nm and below room temperature.

A final word of caution extends to the quality of the lamp. Xe lamps darken quite quickly with age, so long before the lamp should be replaced (say at 1000 h), the far-UV output will have diminished. This makes the head-room for absorbing species much less. Opening the slits can help, but then spectral resolution is lost. Always using two or three cuvettes for setting up these measurements will provide confirming evidence that the measurements are real.

3 APPLICATIONS

3.1 Identification of Protein Secondary Structure

CD spectroscopy has long been used as a simple and direct technique to assess the extent of folding within the protein structure, as well as the type of secondary structure adopted by the protein.¹ In general, proteins with α-helical structure display negative CD signals in the far-UV region at 208 and 222 nm. The second signal is typically used to estimate the α-helix content of a protein. Comparatively, the β-sheet conformation within the protein is characterized by a signal at 215 nm, which may be sinusoidal in nature.

The simplicity of performing this protein secondary structure analysis makes CD spectroscopy useful in monitoring protein conformational changes between wild-type and mutant proteins, as well as in identifying changes in the protein secondary structure upon addition of a bound cofactor such as a metal ion. Several recent examples of applying CD spectroscopy for these purposes are now described.

Yamniuk et al. report the use of CD spectroscopy to measure the structural changes in calcium- and integrin-binding protein upon addition of the divalent metal ions Ca²+ and Mg²+ to the metal-free apoprotein.² They found increases in the α-helical content of the protein of over 30% when either of the metal ions was added to the protein. Futaki et al. showed that the α-helical character of a model peptide could be destabilized by the addition of ferric ion.³ This destabilization of the secondary structure was indicated by suppression of the 222 nm signal upon addition of Fe³+. The authors concluded a possible ferric/ferrous redox control of the helical structure of the peptide.

Dai et al. reported that intermolecular associations within the γ -carboxyglutamate-rich neuroactive peptide conantokin-G could be promoted specifically by the binding of Ca²+ ions to the protein.⁴ The binding of Ca²+ was found to favor an α-helical structure important for intermolecular cysteine–cysteine cross-linking. Interestingly, other divalent metal ions (Mg²+, Zn²+, and Mn²+) bound to the protein with greater increase in α-helical content of the protein as observed in their respective CD spectra as compared to Ca²+ binding, but did not promote the intermolecular cross-linking.

Murphy et al. used CD spectroscopy to monitor the secondary structure of a novel human-derived apyrase (human soluble calcium-activated nucleotidase-1 (hSCAN)) in the presence and absence of calcium ion binding.⁵ Their study indicated that although Ca²+ was essential for enzymatic activity, the binding of Ca²+ was not accompanied by any observable changes in the secondary structure, as the calcium-free protein displayed a β-sheet conformation that was not altered upon Ca²+ addition. By comparison, in a study by Tickler et al.,⁶ CD spectroscopy was used to monitor changes in secondary structure of wild-type amyloid-β peptide (Aβ40) and its methylated histidine imidazole equivalents [Aβ40(Hisτ Me)] and [Aβ40(Hisπ Me)].⁶ This study demonstrated that the binding of Cu²+ to the wild-type peptide and to the π-nitrogen methylated imidazole peptide was accompanied by a conformational shift from random coil to predominantly β-sheet structure.

The increase in β-sheet content in the peptide accelerates the peptides susceptibility to form aggregates.⁷ The further indication that the modified peptide analog methylated at the τ histidine imidazole nitrogen did not change conformation upon Cu²+ binding supports the idea that it is these imidazole nitrogens within the amyloid-β peptide that form bridging structures leading to the oligomerization of the peptide.

The finding that metal binding by a peptide leads to preferred α-helix structure within the peptide was also indicated in a study by Wang et al. using CD spectroscopy to monitor the metal-peptide structure⁸ (Figure 1). The authors prepared a five-residue peptide that models the metal-binding site of the olfactory receptor protein. They found that upon addition of up to one Cu²+ equivalent, the CD signal changes from one characteristic of a random peptide structure to one with the double minima at 209 and 222 nm characteristic of α-helix peptide structure.

Interestingly, α-helicity is very uncommon for a peptide of less than 15 residues, suggesting that binding to the metal ion is essential for creating and maintaining the peptide structure. This structure is thought to require a tetrahedrally coordinated metal ion such as Cu²+, Zn²+, or Ni²+. Using the spectroscopic and structural data for the metal-bound peptide, the authors indicated that the ligation of the metal ion occurs with coordination to one histidinyl nitrogen atom, a cysteinyl sulfur atom, a glutamate oxygen atom, and one solvent molecule that can be replaced by an odorant molecule. This binding is consistent with the 1:1 metal ion:peptide binding ratio observed spectroscopically.

The ability of CD spectroscopy to monitor changes in protein secondary structure is also used to assess the stability of the protein toward thermal or chemical denaturation. This is especially applicable when comparing the structural stability of the protein in the absence and presence of bound metal ions. A number of recent studies have employed CD spectroscopy for this purpose.

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Figure 1 Far-UV CD spectrum of HAKCE peptide, in the absence of metal ions (solid line) and in the presence of 1.0 equivalent of Cu(II) added (dotted line); 20 mM peptide, pH 7.4 potassium phosphate buffer, 298 K. (Wang, J., Luthey-Schulten, Z. A., Suslick, K. S. (2003) Proc. Natl. Acad. Sci. USA 100:3035 – 3039. © 2003 National Academy of Sciences, USA)

Golynskiy et al. used the CD signal at 222 nm characteristic of the α-helical protein conformation to monitor the thermal denaturation of the metalloregulatory protein MntR, a manganese(II)-responsive transcription factor in Bacillus subtilis⁹ (Figure 2). In the metal-free form, the protein displayed a melting temperature of about 67°C. Addition of Mg²+ or Ca²+ had no effect on the melting temperature of the protein. However, the d-group divalent metal ions Mn²+, Co²+, Ni²+, and Cd²+ increased the thermal stability of the protein such that the melting temperature increased by 15 to 30°C upon addition of these metals (Figure 2).

The thermal stability of wild-type peptides and modified analogs in the absence and presence of bound metal ions was also measured using CD spectroscopy in studies by Sissi et al.¹⁰ and by Cox et al.¹¹

In a separate study by Kovári et al., the authors report the use of CD spectroscopy to study the binding of Mg²+ to the DNA repair enzyme dUTPase.¹² This enzyme catalyzes the hydrolysis of uracil triphosphate (UTP) to uracil monophosphate and inorganic pyrophosphate, leading to removal of excess uracil. The Mg²+ ion is an essential cofactor for enzyme activity. CD spectroscopy indicated that Mg²+ binding modulates the conformation of the enzyme allowing for enhanced formation of the enzyme-substrate complex. Binding of the Mg²+ ion and the subsequent binding of the UTP substrate also lead to increased stability of the enzyme toward proteolytic digestion by trypsin.

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Figure 2 Thermal denaturation curves for wild-type MntR (8 μM) monitored by CD spectroscopy at 222 nm: unfolding of apo MntR (open squares) and of MntR in the presence of 1.0 mM Mg²+, Ca²+, Mn²+, Co²+, Ni²+, and Cd²+. Buffer 20 mM HEPES, pH 7.2 at 4°C, 200 mM NaCl, 5% (v/v) glycerol. Pathlength 2 mm. (Reprinted with permission from Golynskiy, Davis, Helmann and Cohen.⁹ © 2005 American Chemical Society)

CD spectroscopy has also provided valuable insight into the chemical stability and chemical denaturation of proteins. A recent study by Rumfeldt et al.¹³ examines the guanidinium-chloride induced denaturation of mutant copper-zinc superoxide dismutases (SODs).¹³ These mutant forms of the Cu, Zn-SOD enzyme are associated with toxic protein aggregation responsible for the pathology of amyotrophic lateral sclerosis. In this study, CD spectroscopy was used in conjunction with tryptophan fluorescence, enzyme activity, and sedimentation experiments to study the mechanism by which the mutated enzyme undergoes chemical denaturation. The authors found that the mutations in the enzyme structure increased the susceptibility of the enzyme to form partially unfolded destabilized monomers, rather than the stable metallated monomer intermediate or native metallated dimer.

Ghosh et al. also used CD spectroscopy to assess the association affinities of peptide coils in the absence and presence of bound Hg²+ or Cd²+.¹⁴ These authors constructed a family of peptides having secondary structures that bind metal ions in a trigonal thiolate coordination environment, even metal ions like Hg²+ and Cd²+ that prefer tetrahedral coordination geometries. Their results indicate increased stability of coiled-coil peptide structures upon addition and binding of the metal ions, and support the hypothesis that favorable tertiary interactions within the protein systems allow for stabilizing the non-preferred coordination geometry of the bound metal ions.

While the study by Ghosh et al.¹⁴ examined the use of CD spectroscopy to elucidate metal ion coordination chemistry as a result of the stabilization of specific secondary structures within the peptide, CD spectroscopy can also directly probe the binding environment and coordination chemistry of metal ions bound to protein ligands. This is the case when functional groups on the peptide chain bind metal ions with the formation of asymmetric metal-ligand clusters due to bond constraints within the peptide structure. This asymmetry in the metal ion environment leads to chiral chromophores centered at the metal ion. This is especially the case when clusters of metal ions are bound within a protein structure by bridging peptide ligands between the metal ions. The chiral electronic transitions that are most often observed in these instances are ligand-to-metal charge transfer (LMCT) transitions most common with d-shell metal ions.

Many d-block metals (Groups 3–12) have important roles in biological systems, both as essential cofactors within a variety of cellular processes, and as interfering species that are toxic to the cell and organism. These metals exist in biological systems as ions that are often bound to proteins and peptides with the exhibition of unique coordination geometries, such that these species become of interest to the inorganic chemist as well as the biological scientist. Because of the ubiquitous nature of the interaction of d-shell metal ions with biological molecules and the inherent sophistication of the metal ion chemistry when bound to the biological molecules, tools that have sought to explain the character of metal-protein binding, including CD spectroscopy, have mainly been applied to the binding of the following metal ions by protein or peptide ligands: Fe²+ and Fe³+, Cu+ and Cu²+, and the Group 12 triad of Zn²+, Cd²+, and Hg²+. The applications of CD spectroscopy to the understanding of metal-protein binding, structure, and reactivity are herein presented with focus on these metal ions, followed by brief descriptions of CD spectral studies of protein molecules binding other metal ions less common to biological environments. Finally, we highlight the use of CD spectroscopy in the determination of speciation in the protein metallothionein.

3.2 CD Spectra of: Complexes of the Group 8–10 Transition Metals

The formation of metal-protein complexes of the transition metals has been an area of intense research, with CD spectroscopy providing essential insights into the structural, electronic and stoichiometric properties of the complexes. The binding of iron ions to proteins has provided the greatest amount of results. As with copper ions, iron complexation with biological ligands is of interest due to iron being both an essential element and a potentially toxic element, as well as the wide range of complexes that iron ions form because of two different stable oxidation states (Fe²+ and Fe³+) and the variety of protein ligands and structural motifs that the iron cations may adopt.

The binding of iron to a model miniaturized protein has been reported by Lombardi et al.¹⁵ The miniaturized protein was constructed based on the tetrahedral tetrathiolate [S-Cys]4 iron-binding site of the rubredoxin protein, and named miniaturized electron transfer protein (METP). The farUV CD spectrum of the apoprotein and of the Fe³+ complex indicate that the protein structure changes from an unordered peptide chain in the metal-free state to an ordered α-helical structure with negative CD bands at 204 nm and 222 nm upon addition of Fe³+. In the near-UV and visible regions of the spectrum, the Fe³+-METP complex displays a strong Cotton effect with signals at 580 nm (+), 500 nm (−), 440 nm (+), 370 nm (+), and 315 nm (−). In the metal-reduced complex of Fe²+-METP, CD signals are observed at 280 nm (+), 305 nm (−), and 320 nm (+). These spectral characteristics, as well as those for the Co²+-METP complex, are similar to the CD spectra of natural and reconstituted rubredoxin, confirming that the same metal-binding environment was maintained in the model system relative to the natural protein.

Multi-thiolate iron-protein clusters are also found in the ferredoxin family of electron transport proteins. In particular, the interconversion between ferredoxin clusters containing three iron atoms and other clusters containing four iron atoms was the subject of research by Jung et al.¹⁶ It was found that the replacement of one cysteine residue central to the metal-binding site with an aspartate residue allowed for the conversion of the redox active site from [4Fe-4S]²+/+ to the [3Fe-4S]+/⁰ cluster. CD spectroscopy is very sensitive to the type of iron-sulfur cluster that is formed within the protein. Native ferredoxin has CD spectra with signals at 310 nm (+), 395 nm (+), 470 nm (+), and 580 nm (+) for the ferric protein, and 360 nm (+), 405 nm (+), 450 nm (−), 510 nm (+), and 580 nm (+) for the ferrous protein. The mutated protein with substitution of a central cysteine → aspartate displayed CD spectra for both the oxidized and reduced forms that were very similar to those for the native ferredoxin protein. This indicates that the mutated protein has iron atoms ligated by only three thiolate cysteine residues, without a distant cysteine providing a fourth thiolate ligand to the cluster. Supporting crystallography data indicate that the aspartate residue itself provides the fourth ligand to the Fe-S cluster through a carboxyl oxygen atom.

CD spectroscopy has also been used to study the metal-binding and reactivity properties of iron- and manganese-containing SODs. These enzymes metabolize the superoxide ion radical c01ie001 into molecular oxygen and hydrogen peroxide. The Fe- and Mn-SOD enzymes are strictly metal specific. Jackson and Brunold¹⁷ show that, despite the fact that Fe replacement of Mn in the Mn-SOD enzyme [(Mn → Fe)-SOD] results in an inactive enzyme, the CD spectrum of wild-type Fe-SOD and (Mn → Fe)-SOD are remarkably similar. This suggests that the destroyed enzyme activity, upon replacement of the metal, does not occur via distortion of the enzyme active site.

CD spectroscopy of the Fe-heme binding site of a protein has also been used to monitor the binding of other metal ions to a protein. Hemopexin is a plasma protein that binds free heme, reducing the extent of iron loss and preventing possible oxidative damage that would be catalyzed by free heme. Hemopexin has also been shown to bind other metal ions. Mauk et al. indirectly studied the binding of Cu²+ and Zn²+ to hemopexin by monitoring the CD Soret signal of the heme iron group within this protein¹⁸ (Figure 3). Addition of up to 18 equivalents of either Cu²+ or Zn²+ altered the CD signal from a bisignate Soret with maximum at 415 nm and minimum at 430 nm, to a positive Cotton signal centered at 420 nm.

These changes were reversed upon addition of excess EDTA. The changes in the CD spectrum caused by Cu²+ or Zn²+ addition to hemopexin suggest structural or electrostatic perturbations in the vicinity of the heme group by the added metal ions. The authors indicate that binding of Cu²+ or Zn²+ to hemopexin may play a role in exchanging ligands to the bound heme group, possibly leading to dissociation of the heme complex from the protein.

In comparison to the large amount of research that has been conducted on copper and iron complexes of biological ligands, similar information with other metal ions is relatively sparse. Vanadium is an essential trace element with a very complex inorganic chemistry, owing to its multiple stable oxidation states and its chelation by a variety of ligand donors. Tasiopolous et al. synthesized and characterized dipeptide complexes of V³+ and oxovanadium(IV) (VO²+) as possible models of vanadium-protein interactions.¹⁹ The dipeptides used in this study all contained glycine as one residue, with alanine, valine, or phenylalanine as the second amino acid. 1,10–phenanthroline was also present as an additional ligand. Part of the characterization of these complexes included CD spectroscopic analysis of the complexes in solution and in the solid state. The authors report that the V³+–dipeptide complexes with phenanthroline had CD spectral signals that were too weak to be measured in solution. However, in the solid state the V³+ complexes displayed visible CD signals at about 740 to 780 nm (+), 480 to 550 nm (−), and 420 to 490 nm (+). By contrast, the VO²+ analogues of these dipeptide complexes had much stronger CD spectral signals that could be measured in solution. CD signals were observed at 725 to 735 nm (+), 470 to 480 nm (−), and 395 to 410 nm (+). In the solid state, the VO²+-dipeptide phenanthroline complexes were characterized by strong signals at 770 to 800 nm (+), 550 to 570 nm (+), and 465 to 485 nm (−). The two lower wavelength signals are inverted in sign relative to the V³+ complexes. These CD signals may be used as a spectral fingerprint of octahedral VO²+ complexes with Namine–Npeptide–Ocarboxylate ligation.

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Figure 3 Effect of Zn²+ binding on the CD spectrum of the hemopexin (Hx)-heme complex: the Hx-heme complex (thick black curve), additions of 3 – 15 equiv of Zn²+ (thin black curve), and addition of 18 equiv of Zn²+ (red curve). The Hx-heme concentration is 8 μM, with 50 mM bisTris buffer and 50 mM sodium chloride (pH 7.0, 25°C). (Reprinted with permission from Mauk, Rosell, Lelj-Garolla, Moore and Mauk.¹⁸ © 2005 American Chemical Society)

The chemistry of cobalt with biological ligands has been an area of increasing research owing to the role of this metal as an essential trace element, for example, as a cofactor in the vitamin B12 molecule, and as a possible mimic of the biological chemistry of other metal ions. Cobalt binds to biological ligands predominantly as the divalent ion, such that its bioinorganic chemistry is expected to be similar to other d-shell divalent metal ions like Zn²+, Cu²+, or Fe²+. The Co²+ ion also displays d–d electronic transitions in the visible region of the spectrum which can be easily monitored by CD spectroscopy during a binding experiment.

Zhu et al. studied the metal-binding properties of copper chaperone proteins using CD spectroscopy by addition of Co²+ as a mimic of copper binding.²⁰ The authors of this study compared the binding of Co²+ to two different copper chaperone proteins, from Tomato copper chaperone for superoxide dismutase (tCCS) and human copper chaperone for superoxide dismutase (hCCS) sources. Both of these proteins are comprised of three structural domains. The tomato protein sequence includes four cysteine residues, two cysteines in each of domains 1 and 3, which were expected to be responsible for metal ion ligation. The human protein has nine cysteine residues with 3, 4, and 2 cysteines in the three respective domains of the protein. The main structural difference between the copper chaperone homologs is that the tomato protein has no cysteine residues in its second domain while the human protein has four cysteines in this domain. Moreover, this domain in hCCS has a great extent of sequence homology with the Zn²+ binding site of Cu, Zn-superoxide dismutases. Thus it is expected that divalent metal ions bind to domain 2 of hCCS with high affinity in a tetrahedral M²+-Scys4 geometry.

Addition of Co²+ to metal-free hCCS initially leads to minimal changes in the CD spectrum, up to addition of one equivalent of the metal ion. Further, addition of a second equivalent of Co²+ results in significant alterations to the CD spectrum, with strong signals appearing at 380 nm (+), 505 nm (−), and 610 nm (+). From these results the authors conclude that the binding of Co²+ to hCCS occurs first with one equivalent binding to the tetra-cysteinyl domain 2 with a symmetric tetrahedral binding geometry, followed by the binding of a second Co²+ to four of the remaining cysteine residues of domains 1 and 3 in a chiral distorted tetrahedral structure.²⁰

Addition of Co²+ to tomato CCS resulted in the immediate formation of strong CD signals at 395 nm (+), 520 nm (−), and 620 nm (+). The spectrum reached saturation after addition of one Co²+ equivalent, at which point the spectrum was almost superimposable to the CD spectrum resulting from addition of two Co²+ equivalents to hCCS. Removal of the domain 1 cysteine residues resulted in much weaker CD signals. These data further indicate that the binding of Co²+ to cysteine residues across domains 1 and 3 of the CCS proteins results in a highly optically active distorted tetrahedral structure with Co²+ bound to four cysteine ligands.

3.3 CD Spectra of Group 11 Metals

Most of the research that has examined the binding of Group 11 metal ions to biological molecules has focused on the binding of copper ions. There are a number of reasons for this, including the range of biological molecules that bind copper ions, the fact that copper is both an essential element acting as a cofactor in numerous enzymatic processes while being potentially toxic at elevated concentrations, the various coordination geometries that copper can adopt, and different binding characteristics between the monovalent and divalent oxidation states of copper ions.

As a general rule, monovalent copper ions bind exclusively to cysteine thiolate ligands of proteins and peptides. The bound Cu+ ions usually adopt the trigonal planar coordination geometry, although digonal binding geometries are sometimes observed, especially with bridging thiolate ligands. As with the Group 12 metal ions, the use of CD spectroscopy to probe the binding of Cu+ to metallothioneins has been a very rich area of research in terms of the biological and inorganic chemistry of the Cu+-thiolate cluster formation. Studies that have examined Cu+ binding to metallothioneins using CD spectroscopy are also discussed within the separate Section (3.6) on metal binding to the metallothioneins.

CD spectroscopy has also been used to monitor Cu+ binding to a copper-transporting protein, the P-type ATPase ATP7B found to be involved in Wilson’s disease, a disorder characterized by over-accumulation of copper in liver cells and other sites²¹ (Figure 4). It was found that the addition of Cu+ (by reduction of added Cu²+ with dithiothreitol) had the most dramatic effect on a peptide derived from the ATPase that contained three cysteine residues, two of which were separated by a single proline residue to give a–CPC–motif. Moreover, the authors of this study examined the nature of the CD spectral bands in order to formulate a hypothesis regarding the speciation of the Cu+-peptide complexes. In this study, four well-resolved signals were detected including a maximum at 270 nm, a double minimum at 290 nm and 320 nm, and another maximum signal centered at 354 nm.

The CD signals in the near-UV to visible region are typically LMCT transitions. The authors also suggest that the higher wavelength signal at 354 nm may be assigned to a spin-forbidden 3d → 4s metal cluster-centered transition that is favored by d¹⁰−d¹⁰ interactions of adjacent Cu+ ions. These transitions would require multiple Cu+ ions binding in close proximity to each other, with bridging thiolate ligation necessary for the assembly of these clusters. The presence of these bridged Cu+-thiolate clusters is also associated with the formation of oligomeric species. The equilibrium between the formation of the monomeric Cu+-thiolate species with only terminal thiolate ligation and formation of the oligomeric bridged Cu+-thiolate clusters is dependent on the stoichiometric ratio of added Cu+ to peptide concentration.

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Figure 4 Changes in the CD spectra of variants of ATP7B, 2K8p and the mutants 1s, 2s and 3s in the region 250 – 400 nm induced by the addition of Cu(II) and DTT (peptide, solid line; peptide + DTT, dot; peptide + Cu(II) 1:1, dash; peptide + Cu(II) + DTT, dash dot). (Reprinted from Myari, Hadjiliadis, Fatemi and Sarkar.²¹ © Elsevier 2004)

Another class of copper transport proteins that have been studied using CD spectroscopy are the copper chaperone proteins. Two copper chaperones, one from B. subtilis²² and another from Enterococcus hirae,²² have recently been examined. The work of Kihlken et al. demonstrated that Cu+ binds to the B. subtilis chaperone protein CopZ in three phases.²¹ The first phase lasts up to a Cu+: protein ratio of 0.5:1, and is characterized by the growth of CD signals at 265 nm (+) and 287 nm (−). The second phase of Cu+ binding to the CopZ protein extends from 0.5 up to 1.0 Cu+ equivalent per protein. This second phase is characterized by a shift and weakening of the 265 nm signal to about 275 nm, and the growth of a broad negative band that is centered at about 335 nm. The final phase of Cu+ binding to the CopZ protein lasts up to a Cu+: protein ratio of 1.5:1. This final phase is characterized by the formation of a derivative-shaped CD signal with a minimum at 265 nm, a maximum at 287 nm, and a sharp isodichroic point at 280 nm. Carrying out the titration in the presence of dithiothreitol, a small molecule-reducing agent that may also act as a sulfur donor ligand, limits the Cu+-CopZ binding mechanism to the first phase only. Along with results from analytical ultracentrifugation and gel-filtration experiments, this CD study for Cu+ binding to CopZ concluded that the binding of Cu+ initially results in the assembly of a dimeric complex with a single metal center. Further addition of Cu+ leads to formation of a second dimeric species with two Cu+ ions bound by four cysteine thiolate sulfur atoms. In this species, two of the cysteine sulfur ligands (one from each peptide subunit) are bridging ligands, leading to closer Cu+−Cu+ interactions. These metal-centered interactions may be responsible for the higher wavelength CD signal that is observed during the second phase of the Cu+ titration. In the third phase of the titration, the dimeric structure is thought to be conserved with an additional Cu+ center within the metal-thiolate cluster.

For Cu+ binding to the CopZ protein from E. hirae, Urvoas et al. also observed the formation of a dimeric species with a stoichiometric ratio of two protein subunits to one Cu+ ion.²³ These authors also report that the dimeric species can be changed to a monomeric complex by the addition of the copper-coordinating molecule glutathione.

As we have seen, the electronic spectra of Cu+ complexes are dominated by LMCT and metal-ligand charge transfer (MLCT) transitions in the near-UV and UV regions only under specific conditions of metal loading and bridging ligation within the metal-protein cluster. No d–d transitions are possible. By comparison, the loss of a d-shell electron to give the Cu²+ ion results in the formation of complexes in which the electronic spectra are dominated by transitions in the visible region.

Ganadu et al. have presented CD spectroscopic evidence for complex formation between Cu²+ and the αβ-crystallin protein.²⁴ This protein is a structural protein of the eye lens that functions as a stress response protein, presumably as a molecular chaperone involved in protecting denatured proteins from aggregation. The chaperone-like activity of αβ-crystallin is enhanced in the presence of the Cu²+ ion, indicating that the protein contains a binding site for this metal ion. The CD spectrum of the αβ-crystallin protein with Cu²+ added is composed of a band at 343 nm, which becomes more enhanced with increased pH. This signal is ascribed to an imidazole N → Cu charge transfer (CT) transition, suggesting that a histidine residue is one of the ligands of the Cu²+ ion. The CD spectrum of the Cu²+-protein complex also shows a strong derivative-shaped signal in the visible region, with a negative band at 550 nm and positive band at about 650 nm. This signal is consistent with a Cotton effect typical for the chiral d–d electronic transitions of optically active Cu²+ complexes.

A further example of the use of CD spectroscopy to monitor Cu²+-protein complex formation is the binding of this ion to the amyloid β-peptide. This peptide is composed of 40 to 43 amino acids residues and is the principal component of the aggregates or plaques associated with neurodegenerative disorders such as Alzheimer’s disease. The ability of this peptide to bind metal ions has been well documented. In fact, it has been shown that Cu²+ and Fe³+ induce the formation of β-amyloid aggregates.²⁵ A study by KowalikJankoska et al.²⁶ sought to elucidate the binding of Cu²+ to the amyloid β-peptide from human and mouse sources. The authors used CD spectroscopy and EPR spectroscopy (see Electron Paramagnetic Resonance (EPR) Spectroscopy) to elucidate Cu²+-peptide complex formation with different fragments of the peptide under a variety of solution conditions. The authors showed that in all cases of Cu²+ binding to the various peptide fragments, the CD spectrum is composed of d–d transitions in the visible region from 480 nm to 670 nm, and LMCT transitions assigned to (i) imidazole N → Cu transitions in the near-UV region from 320 nm to 370 nm, (ii) amide N → Cu transitions Cu transitions at wavelengths from 310 nm to 320 nm, and (iii) amino N → Cu in the region below 300 nm. The intensities and signs of the CD bands, along with complementary data from EPR spectroscopic experiments, are used to arrive at a distribution of Cu²+-amyloid β-peptide species under different conditions of pH and Cu²+ loading.

In a similar way, the binding of Cu²+ to another protein involved in neurodegenerative disease has also been studied using CD spectroscopy. The prion protein is a membrane-associated glycoprotein that undergoes an alteration from a monomeric form to an oligomeric protease-resistant form leading to its toxic effects. It has been suggested that the prion protein plays a role in copper metabolism.²⁷,²⁸ The amino-terminal end of the prion protein is rich in histidine and glycine residues, and is believed to be the metal-binding site of the protein. Specifically, the mammalian prion protein contains a highly conserved region of four repeats of the eight residue sequence –PHGGGWGQ–, the so-called octarepeat sequence. Separate studies by Aronoff-Spencer et al.²⁹ and La Mendola et al.³⁰ have examined the speciation of Cu²+-prion protein complexes using CD spectroscopy, in addition to other spectroscopic techniques. The CD spectra demonstrate the visible region d–d transitions and imidazole N → Cu CT transitions observed in other Cu²+-protein complexes with imidazole and amide ligation. Specifically, in the study by Aronoff-Spencer et al., the data are indicative of a 1:1 stoichiometry between Cu²+ and the aforementioned octarepeat, such that the histidine imidazole provides one ligand to the Cu²+ ion and glycine amide groups provide additional ligation of the metal ion.²⁹

In addition to studying the binding of Cu²+ to known metal-binding proteins, CD spectroscopy has also been used to characterize a novel putative metal-binding protein. Barney et al. report on the isolation of a small (9.9 kiloDalton) protein (SmbP) from the periplasm of the ammonia-oxidizing bacterium Nitrosomonas europaea³¹ (Figure 5). The authors found that this protein was expressed in the bacterium in response to Cu²+ addition to the growth medium, suggesting that this novel protein plays a role in cellular copper metabolism. To characterize the protein, they prepared the metal-free protein and titrated with increasing equivalents of Cu²+.

In the far-UV region, the apoprotein displayed the CD signal characteristic of α-helix secondary structure with double minima at 209 nm and 222 nm. This region of the CD spectrum was unchanged with addition of Cu²+. However, the visible region of the CD spectrum showed dramatic changes during titration of the protein with Cu²+. A positive band was observed at about 490 nm with addition of the first equivalent of the Cu²+ ion, which remained unchanged with further Cu²+ addition. This band is ascribed to a histidine imidazole N → Cu CT transition. Further addition of Cu²+ was accompanied by the formation of a derivative-shaped CD signal with minimum at 580 nm and maximum at 760 nm. These transitions are Cu²+-centered d–d transitions, and their relative energies are suggestive of mixed ligation by nitrogen and oxygen donor ligands. The sequence of this novel protein indicates the absence of any cysteine residues and a large proportion of histidine, aspartate, and glutamate residues, consistent with the suggested ligation of the Cu²+ ions. With further addition of Cu²+ equivalents, the visible region CD signals increase in intensity in a relatively linear manner up to 5 to 6 equivalents of Cu²+ being added. This stoichiometry is further supported by comparable titration experiments monitored by absorbance spectroscopy and EPR spectroscopy. Moreover, a similar titration of the protein with Fe³+ revealed absorbance and CD spectral changes up to a stoichiometry of six Fe³+ equivalents per protein molecule. From these experiments, the authors propose that the novel protein isolated from Nitrosomonas europaea in response to Cu²+ is a metal ion scavenger induced to sequester high concentrations of free Cu²+, and possibly other free metal ions as well.

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Figure 5 Panel (a) shows the UV CD spectra (190 – 250 nm) of apo-SmbP and SmbP (25 μM, pH 7.5, no buffer) with 1 equiv of Cu(II) bound. Spectra for 2 and 5 mol equiv of Cu(II) are not shown because they are virtually identical to the 1 mol equiv of Cu(II). Panel (b) shows the visible CD spectra (400 – 800 nm) of SmbP (250 μM, pH 7.5, no buffer) titrated with increasing equivalents of Cu(II) (0, 0.8, 1.4, 2.7, 3.7, 4.6, and 5.3) from an aqueous solution of CuCl2. Additional mole equivalents of Cu(II) did not result in further changes in the CD spectra. (Reprinted with permission from Barney, LoBrutto and Francisco.³¹ © 2004 American Chemical Society)

In addition to the protein complexes of copper ions, CD spectroscopy has also been used to study metal-protein complex formation of the other Group 11 metals, silver, and gold. The Ag+ ion has been used extensively in metal-protein experiments to act as an analog of Cu+ complex formation. Ag+ is comparable to Cu+ in oxidation state, the types of ligands to which it binds, and often displays equivalent binding geometries to these ligands. Moreover, Ag+ is easier to work with because of its oxidative stability, as it does not have a higher stable oxidation state to which it can be oxidized, as is the case with Cu+. However, the structural properties of Ag+ complexes are not identical to those of Cu+, likely due to the larger ionic radius of Ag+ leading to other preferred binding geometries under certain ligand and stoichiometric conditions. For example, the binding of Ag+ to the metallothioneins results in both similarities and differences in comparison to Cu+ binding to this family of proteins. These specific results will be discussed in the section on applications of CD spectroscopy with metallothioneins (Section 3.6).

Gold complexes with biological ligands present further structural possibilities because of a different preferred oxidation state, the +3 oxidation state. Marcon et al. have employed CD spectroscopy to study complex formation between Au³+ and the abundant serum protein albumin.³²This protein is believed to play a role in extracellular metal ion transport, and may be involved in the exhibited anticancer activity of Au³+-based drugs. The authors determined that bovine serum albumin (BSA) forms very stable complexes with the Au³+ compounds [Au(diethylenetriamine)Cl]²+ and [Au(6-(1,1-dimethylbenzyl)-2,2′-bipyridine)OH]+, such that the metal ion could only be removed from the protein complex by treatment with excess potassium cyanide. The CD spectrum of the BSA complex with the latter of the two Au³+ compounds results in the appearance of visible spectrum signals characteristic of Au³+ complexes bound within a chiral protein environment. These signals include two negative bands, a strong signal at 405 nm and a weaker band at about 340 nm.

3.4 CD Spectra of Complexes involving Group 12 metals

This group consists of the elements zinc, cadmium, and mercury. Each of these metals binds to ligands as divalent metal ions, and usually adopt the tetrahedral binding geometry. These metal ions often bind to thiolate ligands, such as the cysteine sulfur atoms of proteins and polypeptides, although Zn²+ may also bind to the imidazole nitrogen atom of histidine residues. These metal ions are also known to form clusters of multiple metal ions within one protein unit through the chelation by both bridging and terminal peptide residue ligands.

The spectral characteristics of the Group 12 metal ions binding to biological ligands are dominated by the LMCT transitions from the electron-donating ligand atom to the d-shell of the metal atom. These transitions occur in the near-UV region of the spectrum, from 230 nm to 360 nm.

We have previously mentioned that Ghosh et al. have examined the binding of Cd²+ and Hg²+ to peptide coils¹⁴ (Figure 6). They found that the metal ions adopted the unusual trigonal planar binding geometry in order to stabilize the coiled-coil structure of the peptides. This group had previously elucidated the binding geometry of the metal ions to these peptides.³³ In this earlier study, the authors found that the placement of the cysteine ligand along the peptide chain had a profound effect on the metal-protein structure as evidenced by complete inversions of the CD spectra.

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Figure 6 UV-visible titration of TRI L9C into a solution of HgIICl2 at pH 8.5. The data are plotted versus wavelength. The inset shows the titration curve following Δε at 247 nm plotted versus equivalents of peptide added at 247 nm. (Reprinted with permission from Ghosh, Lee, Demeler and Pecoraro.¹⁴ © 2005 American Chemical Society)

The identical energies of the electronic transitions indicate that the coordination geometry of the metal ions, essentially trigonal planar with some trigonal pyramidal character in the Cd²+-bound peptide structure, remained the same regardless of the position of the cysteine ligand. However, the peptide folding around the metal ion was drastically altered upon changing the placement of the cysteine residue, resulting in significant modifications in the constraints placed on the metal-thiolate cluster and different interaction of the chiral center with polarized light accompanied by inversion of the CD spectrum.

The formation of cadmium-protein complexes has also been observed in a protein model of the metal-binding domain of rubredoxin.³⁴ This protein model contains one Cys–X–X–Cys metal-binding motif. CD spectroscopy indicated that addition of the Cd²+ ion to the metal-free protein model resulted in binding of the metal ion with assembly of the protein into a coiled-coil secondary structure. The CD spectrum of the apoprotein model indicated a disordered random structure in the absence of bound metal ions, as evidenced by a major signal at 205 nm and a weak broad band centered at about 220 nm. Binding of Cd²+ led to a far-UV CD signal representative of a coiled α-helix structure in the protein model, with strong negative bands centered at 209 nm and 222 nm, and a thiolate S → Cd charge transfer transition at about 240 nm. Both the LMCT signal and the CD bands representing the α-helix structure of the protein were destroyed upon oxidation of the cysteine thiolate residues, indicating that the reduced thiolate was required to bind the Cd²+ ion leading to stabilization of the α-helix secondary structure of the protein model surrounding the Cd²+-thiolate cluster. Moreover, both CD spectroscopy and UV-visible absorbance spectroscopy were used to monitor the titration of the protein model with increasing relative amounts of Cd²+ ions. It was found that the respective changes in the spectral signals occurred up to a Cd²+: protein stoichiometry of 1:2, suggesting that four chelating cysteine thiolates are required to bind one Cd²+ ion having a tetrahedral binding geometry, such that the Cd²+ ion acts as a bridging atom between two α-helix coiled protein ligands.

Like cadmium, mercury is the subject of significant research owing to its toxicity to living things. Bacterial resistance to mercury poisoning involves a specific periplasmic mercury-binding protein, MerP, and the mercuric reductase enzyme, MerA. Rossy et al. have studied the amino-terminal extension of a mercuric reductase that resembles the Hg²+-binding motif of a MerP protein.³⁵ Using CD spectroscopy to monitor Hg²+ addition to the MerA amino-terminal extension, the authors noted that Hg²+ ion binding occurs with three main characteristics: an increased intensity and blue-shifting of a signal at 265 nm in the metal-free peptide to about 260 nm upon Hg²+ addition, the development of a negative CD signal centered at about 290 nm, and a sharp isodichroic point at 270 nm. Changes in the CD spectrum were observed for Hg²+ addition up to one mole equivalent with respect to the MerA amino-terminal model peptide, indicating only one metal-binding site within the peptide. The isodichroic point indicates the existence of only two possible states for the peptide, these being the metal-free and Hg²+-bound forms. Moreover, the authors report that the far-UV CD signal did not change upon Hg²+ addition, indicating no change in the secondary structure of the peptide with metal binding. The authors propose that the metal-free peptide adopts a structure such that it is poised to bind the metal ion. This may be a critical property of a protein conferring mercury resistance to an organism.

Modeling of the metal-binding characteristics of the mercury-resistance proteins was also the subject of a study by DeSilva et al.³⁶ The authors constructed 18-residue peptides with metal-binding sites consisting of two cysteine residues and two alanine residues, with metal-binding site sequences as –CCAA–, –CACA–, or –CAAC–. The binding of each of the Group 12 metal ions, as well as Group 11 metal ions Cu+ and Ag+, were monitored by CD spectroscopy. The authors found that Hg²+ bound the strongest of any of the metal ions to the peptide containing the –CAAC–motif, and was the only metal ion to bind to the –CCAA–containing peptide. It is thought that the fact that Hg²+ can adopt a range of geometries, specifically the linear digonal coordination, allows it to bind to the peptide with the neighboring cysteine residues.

Similar metal-binding motifs are common in various proteins and peptides with metal-binding capabilities. Suzuki et al. examined the metal-binding characteristics of one such protein, the heavy metal-binding protein Cdl19 from Arabidopsis.³⁷ The authors prepared the 225 amino acid residue amino-terminal peptide containing the –CXXC–metal-binding motif. Addition of Cd²+ to the cysteine-containing peptide was accompanied by significant changes in the CD spectrum. These changes were not observed if the cysteine residues were replaced with glycine. Similar spectral changes were also observed upon addition of Cu²+ and Hg²+, consistent with chelation of the metal ions by the cysteine

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