1 ELECTRON PARAMAGNETIC RESONANCE

ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY OF METALLOPROTEINS

Short title:

EPR OF METALLOPROTEINS

Author:

R. Cammack

Affiliation:

King's College London

Full address:

Division of Biomolecular Sciences, King's College London, Campden Hill Road, London W8 7AH

Tel:

+71 333 4264

Fax:

+71 333 4141

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Table of Contents Electron paramagnetic resonance spectroscopy OF METALLOPROTEINS1 Short title: EPR OF METALLOPROTEINS .......................................... 1 Table of Contents ............................................................................ 2 Abbreviations and Symbols: ............................................................ 4 1. Introduction ................................................................................. 5 1.1. Systems that can be studied by EPR. ....................................... 7 1.2. Principle.................................................................................... 7 1.3. Detectable Valence states .......................................................... 8 1.4. Factors affecting the line shape of the EPR spectrum................. 8 2. Materials and Methods ................................................................. 11 2.1. Spectrometers. .......................................................................... 11 2.1.1. Computers ............................................................................. 11 2.1.2. Temperature control ............................................................... 12 2.2. Cell holders ............................................................................... 12 2.2.1. Cells for aqueous samples ...................................................... 12 2.2.2. Frozen solutions. .................................................................... 13 2.3. Sample preparation ................................................................... 14 2.3.1. Concentration methods. ......................................................... 15 2.3.2. Oxidation and reduction treatments ....................................... 15 2.3.3. Isotope substitutions. ............................................................. 16 2.3.4. Preparation of oriented samples ............................................. 16 3. Experimental ............................................................................... 17 3.1. Measurement of the EPR spectrum............................................ 17 4. Information from EPR spectra: ..................................................... 18 4.1. Identification ............................................................................. 18 4.2. Resolution of spectra of impure biological extracts..................... 18 4.3. Quantitation ............................................................................. 19 4.4. Microwave power saturation ...................................................... 19 4.5. Coordination geometry .............................................................. 20 4.6. Environment of the metal ion .................................................... 20 4.7. Redox potentials........................................................................ 21 5. Conclusions ................................................................................. 21 Acknowledgements........................................................................... 22 References ....................................................................................... 22 2

Figure Captions ............................................................................... 25

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Abbreviations and Symbols: c.w.

continuous-wave

EDTA

ethylenediamine tetra-acetate

ENDOR

electron-nuclear double resonance

EPR

electron paramagnetic resonance

ESEEM

electron spin-echo envelope modulation

ESR

electron spin resonance

GHz

gigahertz

kHz

kilohertz

mT

millitesla

NMR

nuclear magnetic resonance

A

hyperfine coupling constant

B0

magnetic flux density (magnetic field)

g

spectroscopic g-factor

h

Planck's constant

I

nuclear spin

mM, M

millimolar, micromolar

ms

electron spin quantum number

S

electron spin

T1,T2

electron spin-lattice and spin-spin relaxation time



Bohr magneton



microwave frequency





Bohr magneton

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1.

Introduction

Electron paramagnetic resonance (EPR), or electron spin resonance (ESR) spectroscopy, is a technique for studying paramagnetic materials, the molecules of which contain unpaired electrons. These comprise organic free radicals and transition metal ions. The biological significance of these rare species is that they often occur at the active sites of enzymes, and the electron-transfer systems of bioenergetics. Manganese ions are of special significance in molecular biology because they can often bind to nucleotides and nucleic acids in a similar way to magnesium, and serve as probes of their environment. There are at least three main areas of interest for biological EPR. The first of these is to examine the naturally-occurring transition metal ions and radicals in a sample, and learn about their function and environment. The second is to use artificially-introduced radicals or spin-labels as probes of the environment and dynamics of a particular biological species such as a protein or lipid. The third is to use spin-traps to identify the short-lived radicals produced during biological processes, such as reactions with oxygen. Each of these is a large area, but there are some areas of technique which overlap. For example, they all use the same type of spectrometer. and there are similar problems of sensitivity and contamination. This short review covers the first of these objectives, with particular reference to transition metal ions. It is intended to provide practical guidance to biochemists and molecular biologists who need to make EPR measurements of their material. Mention will be made of the problems of obtaining samples in the right oxidation state and in sufficient concentration to provide recognizable EPR signals; and of resolving the complex signals which arise in biological material. More details about the operation of the EPR spectrometer, and the theory of transition-metal EPR, are provided elsewhere [1-3]. Because the magnetic moment of the electron is hundreds of times larger than those of nuclei such as 1H, EPR spectroscopy is in principle much more sensitive than NMR. EPR can be used to measure transition ions in relatively low concentrations. It can be applied, for example, to a metalloenzyme, or to cell extracts containing that enzyme, or even, if it is present in sufficient amounts, to whole cells. In order to measure transition metals ions by EPR, it is often necessary to use low temperatures. This is a consequence of the extremely rapid electron-spin relaxation rates. As a result, the sample must be in the frozen state, which prevents most measurements of mobility and kinetics. Only free radicals (including spinlabels and spin-traps) and a few transition ions, notably manganese, are readily detected at room temperature. However EPR is a non-destructive method, so the sample can be re-used if it survives freezing. Although it can be used quantitatively, EPR is not a general method to detect the total amount of a metal ion in solution. Standard methods such as atomic absorption spectrophotometry are adequate for this. The value of EPR lies in its selectivity. It can observe individual chemical forms of the 5

metal or radical, even in quite complex mixtures, and can provide information such as their valence state and ligands. As in NMR, spin-spin interactions between the electron and other electrons or nuclei, can be informative about structure. By studying changes that occur in the EPR spectrum of one of these species as a result of biological reactions, we can learn about its function. 1.1.

Systems that can be studied by EPR.

Paramagnetic transition metal ions of biological interest, which can readily be detected by EPR spectroscopy, are iron, copper, manganese, molybdenum, and, more rarely, vanadium, cobalt and nickel. The measurable oxidation states of these elements, and their isotopes that have nuclear spins, are summarized in Table 1. They are sometimes combined in clusters, such as in iron-sulphur proteins, which are also paramagnetic. Another source of paramagnetism in electron-transfer systems arises from free radicals of organic molecules such as flavins and quinones. Radicals may also be induced by reactions of reduced organic matter with oxygen, and by irradiation. 1.2.

Principle

In most compounds the electrons are paired, and therefore diamagnetic. Only those molecules that have unpaired electrons are detected. The electron has an associated spin, S = 12 which gives rise to a magnetic moment, . EPR is therefore a magnetic resonance phenomenon, like nuclear magnetic resonance. It relies on the splitting of the energy levels of the electron states, ms = ±12 , by an applied magnetic field. Resonant absorption results from the excitation of electrons from the lower to the higher energy level, by interaction with microwave radiation. A conventional continuous-wave (c.w.) EPR spectrum is obtained by measuring the microwave absorption at fixed frequency, while continuously scanning the applied magnetic field, B0. The position of resonance of a paramagnet is defined by the g-factor, derived from the equation: h= g B0. or

g

=

h

 B0 .

 = 71.448 B . 0

if  is in GHz, B0. in mT. It can be seen from this equation that the higher the g-factor, the lower the resonant magnetic field. A typical EPR measurement would involve absorption of microwaves with a frequency of 9 GHz in a magnetic field of about 320 millitesla (3200 gauss). This is close to the free-electron g-factor of 2.0023. Transition metal ions may have g-factors that are substantially different from 2, because of interactions between the electron spin and the orbitals. The gfactor is treated as a characteristic parameter of the particular spin system. It may be used in a diagnostic way. analogous to the chemical shift in an NMR spectrum.

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The EPR spectrum is conventionally presented as the first derivative of the microwave absorption. This derivative spectrum is produced by the detection system, which employs magnetic field modulation to imporove the signal:noise. Therefore the typical EPR spectrum has features both above and below the baseline. A simple spectrum, without hyperfine splittings, only crosses the baseline once. 1.3.

Detectable Valence states In general, EPR is readily detected in systems with a single unpaired 1

electron, such as Cu(II) (electron spin S= 2 ), or an odd number of electrons 5

such as Mn(II) (S = 2 ). Oxidation or reduction of a paramagnetic ion will change it either into a form which has zero net spin, such as Cu(I) or lowspin Fe(II), or into one which has an even spin, such as high-spin Fe(II) (S = 2). As a result, EPR is sensitive to the oxidation state of the transition ion. 1.4.

Factors affecting the line shape of the EPR spectrum

The form and characteristics of the EPR spectrum are influenced by a number of effects. Often quite subtle differences in the coordination state of a transition ion can drastically affect the shape of the EPR spectrum. Space does not permit a description of the quantum-mechanical basis of these effects, which are described in standard texts [2]. They will only be mentioned in terms of the practical information that can be derived from them in a biochemical system. These include l Spin-orbit coupling, which affects the g-factor. l Hyperfine coupling, A, with the metal nucleus, and superhyperfine

couplings to ligand nuclei, which give rise to broadening or splitting of the spectrum into (2I +1) lines, where I is the spin quantum number of the nucleus.

l Zero-field splittings in multi-electron ions, which can cause extreme shifts

in the apparent g-factor.

l Electron spin-lattice relaxation rate, T1, which affects the temperature-

dependence of the EPR signal.

l Electron spin-spin relaxation rate, T2, which affects the line width of the

spectral lines.

l Interaction with distant electron spins. The electrons interact with each

other by exchange coupling and dipolar coupling. Strong exchange coupling between two adjacent spins will completely alter the form of the spectrum, or cause it to disappear completely. Weaker interactions can be observed between spins at distances up to 2 nm, as splittings or broadening of the constituent spectra, or an increased relaxation.

l Anisotropy is an important concept which means that all of these effects on

the EPR resonance vary with the direction in which the B0 field is applied to the molecule. Since the molecules in a typical sample are oriented

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randomly, the resultant spectrum is the average of the spectra of all orientations. l Strain, which results from distortions of the local geometry of the metal

centres. The statistical distribution of strain, together with the way in which it influences the g-factor, A-values and relaxation, causes further broadening. All of these factors affect the form of the EPR spectrum, and the conditions in which it is detected. Examples are shown in Fig. 1. The 1

spectrum of Mo(V) (Fig. 1c) is a simple S = 2 system, with a typical g-factor slightly less than 2. The spectrum of Ni(III) (Fig. 1d) has a g-factor greater than 2, with pronounced g-factor anisotropy. Manganese (Fig. 1e) has a gfactor close to 2.0, but the spectrum is split into six lines, by hyperfine interaction with the 55Mn nucleus. The spectrum of Cu(II) (Fig. 1b) has a typical axial lineshape, with hyperfine splitting of g into four lines. The spectra of iron are more complex, because this multi-electron ion can take up two different spin states., depending on the ligands around it and their geometry. High-spin Fe(III) has all of its five 3d electrons in 5

separate orbitals (S = 2 ). Low-spin iron has four of its 3d electrons paired, 1

so that the net spin of the ion S = 2 . The resulting EPR spectra are quite distinct. The g-factors are spread over a very wide range due to zero-field splittings, which are usually highly anisotropic. In Fig. 1(i) the spectrum of a high-spin ferrihaem is axial, with maximum and minimum g-factors of g = 2, g = 6. These values correspond to the cases where the applied magnetic field lies parallel and perpendicular to the normal to the haem plane, respectively. The signal at g = 4.3 (Fig. 1h) is typical of highly distorted Fe(III) centres and is commonly seen in spectra of biological systems. The spectrum of low-spin Fe(III) (Fig. 1g), as observed in many oxidized cytochromes, has g-factors less than 4. When several metal ions form a cluster, their electron spins are coupled together, forming a new spin system. An example is the [2Fe-2S] iron-sulphur cluster in a ferredoxin (Fig 1f). In the reduced protein, ferric 5

1

ion (S = 2 ) and ferrous ion (S = 2) couple, to give a net spin of S = 2 , which gives rise to the EPR spectrum. Spectra of such clusters often have irregular properties such as unusual g-factors, and a strong dependence on the temperature of measurement. It can be seen that the form of the spectrum of any particular metal ion depends on the type of metal ion, its valence state and on the environment.

2.

Materials and Methods

EPR spectroscopy can readily be applied to purified proteins, and in favourable cases even to whole cells. 2.1.

Spectrometers.

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At present only two manufacturers, Bruker Analytische Messtechnik GMBH (Germany) and Jeol Instrument Company (Japan), produce commercial c.w. EPR spectrometers. There are still a large number of instruments made by Varian (USA) in operation. All of them use the same general principle. 2.1.1.

Computers

EPR spectrometers are now provided with a computer system to assist in the setting up and running of the instrument. Specific applications include: l Signal averaging: to enhance the signal:noise ratio of spectra of dilute

samples.

l Spectral subtractions: to resolve complex spectra containing multiple

overlapping signals

l Storage of spectra on disc: for comparison and replotting. l Simulation of spectra: The parameters of a spectrum may be derived by

computer simulation, in which the shape of the EPR spectrum is calculated from assumed values for g, A, and a general line shape function. The values of these parameters are varied, sometimes iteratively, to find the best fit to the experimental spectrum. This is the most exact way of estimating the values of these parameters, and of resolving spectra which contain a number of components. 2.1.2.

Temperature control

For measurements at low temperatures, the spectrometer is fitted with a cryostat. Although immersion cryostats that contain liquid nitrogen (77K) or liquid helium (4.2 K) are simple to operate, they provide only a fixed temperature. Flow cryostats are more flexible, and are now most widely used. Liquid nitrogen is satisfactory for most studies of copper, molybdenum or manganese ions. Liquid helium is necessary to achieve temperatures below 77K which are required for iron-containing proteins. 2.2.

Cell holders

Sample holders are normally made of pure quartz; borosilicate glass contains metallic contaminants which give spurious EPR signals. Some plastics may also be used; it is best to check these for EPR signals before use. 2.2.1.

Cells for aqueous samples

Liquid water and other polar solvents are "lossy". They attenuate the microwave power so much that the instrument cannot be tuned. This can be minimized by the appropriate shape and position of the sample holder in the cavity. In the standard rectangular cavity, the optimum configuration is a flat cell (thickness < 0.5mm), positioned across the centre of the cavity . In a cylindrical cavity the optimum is a capillary aligned with the axis of the cavity. The exact position is adjusted while the cavity is tuned, to minimize microwave loss. Avoid using high microwave power, which can overheat the sample.

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2.2.2.

Frozen solutions.

Sample tubes for solid samples are typically made from quartz tubing of 3 mm internal diameter, 150 mm length, and sealed at one end. The signal size will be proportional to the square of the internal diameter, so for quantitative comparisons the internal dimensions of the tubes should be identical. The sensitive region of the cavity is only 10 - 20 mm, so if the sample is correctly positioned, the minimum volume required for optimum signal is about 100 l. Tubes can be filled, and additions can be made, by using a syringe with a long needle. Additions of substrates, etc., to the sample are made directly into the EPR tube. The samples are thoroughly mixed by stirring up and down with a stainless steel wire, formed into a loop at the end. Any bubbles formed can be removed by vigorously shaking the tube, like a clinical thermometer. Samples are frozen in liquid nitrogen. To avoid breakage, hold the tube at the surface and freeze carefully and slowly, starting from the bottom. Faster freezing may be achieved by plunging the tubes into a small container of 2-methylbutane or methanol, pre-cooled in liquid nitrogen. Store the sample tubes in a container of liquid nitrogen, but above the surface, otherwise oxygen will condense inside. To thaw the sample tubes, hold them in the air for a few seconds to allow any liquid gases to evaporate, then plunge them into warm water. ** WARNING** Aqueous samples in quartz tubes that have been frozen in liquid nitrogen can easily break when they are warmed up, sometimes explosively. Safety spectacles should be worn while handling them. Some commonly-used buffers, particularly phosphate and Tris, can drastically change their pH when freezing. It is better to use zwitterionic buffers which do not suffer from this effect. 2.3.

Sample preparation

The minimum concentrations to detect a paramagnetic species depend on the form and characteristics of its spectrum. For a narrow-line spectrum such as a free radical, or manganese ions in solution, the minimum concentration that may be detected may be as low as 0.1 M, whereas a species with a very broad spectrum might need 50 M. Concentrations to obtain a well-defined spectrum should be ten times greater. In practice another limitation to the amount that can be detected is contamination, either in the form of background signals from the cavity and cryostat, or by paramagnetic impurities in the sample. The former can be minimized by cleaning or subtracted out with the computer, while the latter can be minimized by appropriate purification. Mn2+ ions are present in most cell extracts, and give a strong six-line spectrum around g = 2, which can be substantially broadened out by the addition of 2mM EDTA. Occasionally, a speck of dust or other contamination in the sample holder can cause spurious signals in the spectrum. For this reason, and because the conditions for preparation may prove not to be optimum, it is 10

prudent to prepare duplicate samples if possible. On the basis of the preliminary measurements it may be decided to alter the conditions of preparation. Sometimes it may be possible to add further reagents to the sample, but in other cases the treatment is irreversible. It is always safest to keep some of the material in reserve for further preparations, 2.3.1.

Concentration methods.

Although EPR is more sensitive than NMR, it still requires much higher concentrations of protein than are used in conventional biochemical procedures such as enzyme assays. For good spectra, samples should be as concentrated as possible. Fortunately, only small volumes are needed. For membrane preparations such as mitochondria, samples should be pelleted in a centrifuge and resuspended in the minimum volume. For solutions there are various concentrating methods, perhaps the most convenient for proteins being small-scale centrifugal concentrators that use an ultrafiltration membrane. A cheap alternative, if the solution is not too salty, is to dry the sample down under a stream of nitrogen. 2.3.2.

Oxidation and reduction treatments

Often samples must be oxidized or reduced, to bring the transition metal ion into the paramagnetic oxidation state. Potassium hexacyanoferrate (III) (ferricyanide) is a commonly-used general-purpose oxidizing agent, while ascorbate and sodium dithionite are used as reducing agents. Dithionite reacts spontaneously with oxygen, forming acid bisulphite and oxygen radicals, so it should be added as a freshly-made solution, prepared in a vessel flushed with nitrogen or argon. In an open EPR tube, the contents can be protected from reoxidation by "blanketing" the surface with argon through a syringe needle. The dithionite is injected directly into the solution with a long-needle microlitre syringe. Normally an excess reductant is added, say 2 mM, but more precise reduction may be made either by stoichiometric addition of reducing agent, or by mediator titration [4]. These additions of substrates are relatively slow, and the sample will generally have reached equilibrium before the measurement is made. For kinetic studies it is necessary to use rapid-freeze techniques [5]. 2.3.3.

Isotope substitutions.

In order to identify the metal in a paramagnetic centre, or the nuclei in its vicinity, it is often desirable to replace the metal or other atoms by a stable isotope with a different nuclear spin. This will cause a predictable change in the spectrum due to hyperfine or superhyperfine interactions. 1 For example, to determine if a signal is due to iron, 57Fe (I = 2 ) might be

substituted for the naturally abundant 56Fe (I = 0). Unlike radioactive tracer experiments, where only small amounts of the isotope are introduced, the stable isotope must account for most of the element in the product. This can be done by removal and replacement of the metal, using the techniques of protein chemistry. Alternatively the organism may be grown on a medium containing the appropriate isotope. This is not usually feasible in a mammal for 57Fe, 11

though it has been done in a cow, for 95Mo [6]. It is easier to use a microorganism; an over-producing strain is best, provided it inserts the metal correctly. 2.3.4.

Preparation of oriented samples

Since EPR spectra of most paramagnetic ions are anisotropic it follows that if the molecules in a sample can be aligned, the spectrum will change as the sample is rotated in the EPR cavity. Such alignment is found in crystals, and if this can be achieved, it is possible to determine the direction of the principal axes of g and A relative to the crystal axes. This is rarely practicable in biological systems. However it is found that the proteins, such as cytochromes, in biological membranes, such as mitochondria, are oriented relative to the membrane plane. These membranes can be stacked into an EPR sample by centrifugation onto a Mylar sheet and careful drying down under controlled humidity. In this way the direction of the haem planes in membrane plane has been identified [7].

3.

Experimental

3.1.

Measurement of the EPR spectrum

As may be seen from the number of parameters that affect the form of the EPR spectrum, it is necessary to manipulate the EPR spectrometer conditions to optimize the spectrum of a particular component. Often the choice of correct conditions can make the difference between an almost imperceptible signal, and a strong one. The most important parameters that affect the EPR spectrum are: 3.1.1. Temperature: The optimum temperature for any particular measurement is determined by trial and error. If the temperature is too high, the spectrum disappears due to relaxation broadening. Generally the sensitivity improves as the temperature is decreased, until microwave power saturation sets in. 3.1.2. Microwave power: the signal:noise ratio of the spectrum improves with increased power, at least up to 20 milliwatts. At low temperatures and high power, the spectrum may be diminished and distorted by microwave power saturation. 3.1.3. Magnetic field range: usually defined by centre field and sweep width. Obviously these must be wide enough to cover the spectrum of interest. With unknown samples it is best to start with a wide sweep, say 50 - 450 mT, then narrow down on features of interest. 3.1.4. Modulation amplitude: effectively a bandwidth defining the narrowest line that can be obtained. To avoid distortion, this should be one-third of the narrowest feature to be observed. Typically set to 1 mT (10 gauss) for general broad scans. 3.1.5. Scan time and time constant: These are adjusted to give reasonable signal:noise, a longer time constant giving less noise but requiring a slower scan. For very noisy signals it is preferable to take multiple scans and signal-average.

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The spectrum of a typical cell extract or impure preparation will contain signals from free radicals at g = 2, ferric iron around g = 4.3, and manganese, centred around g = 2. These may be species of interest but often derive from contaminants. The species of interest may be identified for example by oxidation or reduction with specific substrates.

4.

Information from EPR spectra:

EPR spectroscopy is used to identify and measure specific forms of metal ions and radicals in biological materials. 4.1.

Identification

In practice, regardless of the complications of coordination geometry, the characteristic line shape and temperature dependence of the EPR spectrum may often be used in a diagnostic way, to identify a metal centre in a protein. Examples are given in Fig. 1. If the nucleus of the transition ion has a spin, such as 55Mn (nuclear 5

3

spin I = 2 ), or 63Cu (I = 2 ), the hyperfine splitting can aid the identification of EPR signals, particularly in complex spectra with several components. 4.2.

Resolution of spectra of impure biological extracts

It is possible to vary the conditions of sample preparation and instrumental parameters so as to emphasize the spectra of the individual components. This can be done by adjusting l the redox potential; l the addition of specific substrates; l the temperature of measurement; l other instrument parameters, e.g modulation amplitude

4.3.

Quantitation

A distinguishing feature of EPR spectroscopy is that the absolute intensity of an spectrum, regardless of its shape, is a direct measure of the number of electron spins in the sample. This value is obtained from the spectrum by integrating it twice. It is important that that the whole spectrum is measured in isolation, with flat baseline on either side. Before integration, corrections are made for baseline position and slope (Fig. 2). It is possible to estimate the concentration by comparing the double integral of the spectrum with that of a sample of a paramagnet of known concentration, such as a frozen solution of 1 mM copper + 10 mM EDTA. If both samples are of similar geometry and measured under the same conditions, the double integrals of the spectra may be compared directly. This method is only applicable under restricted conditions, namely that the 1

spectrum corresponds to an integral spin (S = 2 ), and is not saturated with microwave power. Practical aspects of quantitative EPR, and the effects of various instrumental parameters, have been discussed in detail by Randolph [8]. 4.4.

Microwave power saturation 13

This occurs when the electron spins cannot dissipate the microwave energy sufficiently rapidly. The rate of energy dissipation, in the form of heat, to the surroundings, is defined by the electron spin-lattice relaxation time, T1. It leads to a decreased signal amplitude and distortion of the spectrum. Power saturation is much more pronounced at lower temperatures, as T1 is strongly temperature-dependent. Power saturation may be observed experimentally by measuring the intensity of the EPR signal as a function of the applied microwave power. Under non-saturating conditions the signal should be proportional to the square root of the power, but this will decrease at higher power as the signal is saturated. The data may be plotted as shown in Fig. 3. The power saturation is characterized by a power for half-saturation, P1/2, which can be derived from the plot. Although in principle this parameter is related to the product T1T2, its most practical application lies in observation of changes of electron-spin relaxation as a result of spin-spin interactions [9]. 4.5.

Coordination geometry

The dependence of g and A on the direction of the BO field, should in principle provide information about the coordination geometry around a metal centre. Unfortunately it is often misleading. For example, the spectrum of a Cu(II) protein such as plastocyanin indicates axial symmetry, which might be taken to imply square-planar or pyramidal geometry; in fact the coordination is a highly distorted tetrahedron. 4.6.

Environment of the metal ion

The identity of ligands may be ascertained from the superhyperfine interaction with the spins of ligand nuclei, notably 14N (I = 1). These nuclear spins may cause splitting or broadening of the EPR spectrum. This is particularly noticeable when the spectra are compared of samples with 1 different isotopes having different nuclear spins, for example 15N (I = 2 )).

Similarly, interactions with exchangeable protons may be detected by a decrease in the line width on exchanging into 2H2O. The effects may be seen in spectra with narrow line widths, when a number of ligand nuclei may be identified. A good example is the measurements of the coordination of the molybdenum in xanthine oxidase by Bray [10]. They are less easily detected in spectra of broad species such as most iron or copper complexes. In order to resolve very small hyperfine splittings it is necessary to use more specialist techniques such as electron-nuclear double resonance (ENDOR) or electron spin-echo envelope modulation (ESEEM). 4.7.

Redox potentials

The midpoint oxidation-reduction potentials of individual components can be resolved by means of the mediator titration technique. This method has been used extensively for membrane-bound electron transport components. It involves adjustment of a solution of the material to a known redox potential, then measurement of the extent of oxidation or reduction from the amplitude of its EPR signal. Often this means removing a sample and freezing it for spectroscopic measurements. A vessel for this purpose,

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and details of sample preparation, have been described by Dutton [4]. From a series of samples poised at known redox potentials, it is possible to plot a graph from which the midpoint potential can be estimated (Fig. 4). The advantage of this method is that the species does not have to be pure, so long as some portion of its EPR spectrum is detectable, and indeed the midpoint potentials of several species can be estimated in the same set of samples.

5.

Conclusions

EPR can be used to identify and quantify specific forms of metal ions in biological systems. Important considerations are that the samples are prepared in sufficiently high concentrations, under the appropriate redox state, and avoiding compounds that give interfering signals. The choice of the measurement temperature and running conditions, is important for acquiring good spectra. EPR offers the ability to determine the effects of spin-spin interactions with ligand nuclei and other unpaired electrons, which complicate the spectra but offer the possibility of obtaining additional information.

Acknowledgements I thank Dr E.C. Hatchikian and Dr R.C. Bray for some of the samples used in Fig. 1. This work was supported by grants from the U.K. Science and Engineering Research Council.

References 1. Czoch, R. & Francik, A. (1989) Instrumantal effects in homodyne electron paramagnetic resonance spectrometers, Ellis Horwood. 2. JE Wertz & JR Bolton, (1986) Electron spin resonance, McGraw-Hill, New York. 3. Gibson, J.F. (1987) Electron paramagnetic resonance in metalloproteins in Clark,R.J.H.,"Spectroscopy of inorganic-based materials" (Ed. Hester,R.) pp. 333-406, Wiley, Chichester. 4. Dutton, P.L. (1978) Redox potentiometry: determination of midpoint potentials of oxidation-reduction components of biological electron-transfer systems, in "Methods in Enzymology: Biomembranes ", vol. 54 (Ed. S. Fleischer & Packer L. ), pp. 411-435 Academic Press, New York. 5. Ballou, D.P. (1978) Freeze-quench and chemical-quench techniques, in "Methods in Enzymology: Biomembranes ", vol. 54 (Ed. S. Fleischer & Packer L. ), pp. 85-93, Academic Press, New York. 6.

George, G.N. & Bray, R.C. (1988) Studies by electron paramagnetic resonance spectroscopy of xanthine oxidase enriched with molybdenum-95 and with molybdenum-97. Biochemistry, 27, 3603-3610.

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7. Poole, R.K., Blum, H., Scott, R.I., Collinge, A. & Ohnishi, T. (1980) The orientation of cytochromes in membrane multilayers prepared from aerobically grown Escherichia coli K12. J. Gen. Microbiol. 119, 145-154. 8. Randolph, M.L. Quantitative considerations in electron spin resonance studies of biological materials (1972) in "Biological applications of electron spin resonance", Swartz, H.M., Bolton, J.R. & Borg, D.c., eds. WileyInterscience, New York. 9. Rupp, H., Rao, K.K., Hall, D.O. & Cammack, R. (1978) Electron Spin Relaxation of Iron-sulphur Proteins Studied by Microwave Power Saturation. Biochim. Biophys. Acta, 537, 255-269. 10. Bray, R.C. (1988) The inorganic biochemistry of molybdoenzymes. Quart. Rev. Biophys., 21, 299-330.

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Table 1: Properties of transition ions relevant to biological systems. Metal ion

Other Paramagnetic oxidation states states

Isotopes

Nuclear Spin

Vanadium

VO(IV)*

V(V), V(III)

51V

7 2

Manganese

Mn(II)*,Mn(IV)

Mn(III)

55Mn

5 2

Iron

Fe(III)*

Fe(II)*

57Fe

1 2

Cobalt

Co(II)*

Co(I)*, Co(III)*

59Co

7 2

Nickel

Ni(I), Ni(III)*

Ni(II)*

61Ni

3 2

Copper

Cu(II)

Cu(I)

63Cu

3 2

65Cu

Molybdenum

Mo(V)*

Mo(IV)*,

95Mo

Mo(VI)*

97Mo

3 2 5 2 5 2

Note - Other oxidation states and isotopes exist for these elements. * denotes commonly-occurring states on biological material

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% Natural Abundance of isotope

99.76 100 2.19 100

1.134 69.09 30.91 15.72 9.46

Figure Captions Fig. 1. Examples of EPR spectra, plotted on a wide field scan to emphasise the range of g-factors of the spectra of transition-metals in proteins. The samples, and temperatures of measurement, were (a) an organic radical, at g=2.003 (room temperature); (b) Cu(II) in superoxide dismutase (77K); (c) the desulpho-inhibited molybdenum(V) signal from milk xanthine oxidase (150K) (the field scan is too wide to reveal the rhombic lineshape of this spectrum); (d) nickel(III) in hydrogenase from Desulfovibrio gigas; (80K); (e) Mn(II) ions in solution (room temperature); (f) reduced [2Fe-2S] cluster in spinach ferredoxin (24K); (g) low-spin Fe(III) in metmyoglobin azide (30K); (h) highspin Fe(III) in D. gigas rubredoxin (12K); (i) high-spin Fe(III) in methaemoglobin (10K). Fig. 2. Method of double integration of a spectrum of an S = 12 species. The spectrum, obtained as a series of digital values on the computer, is first levelled by subtracting a sloping baseline. Next the first integral is obtained by numerical summation of the points. A second correction is made to level the spectrum again and the second integal is taken. The value shown is proportional to the concentration of spins in the sample. Fig. 3. Example of a power saturation curve. The amplitude of an EPR signal is measured with varying microwave power, keeping other parameters, especially temperature, constant. The value P1/2 is a measure of the degree of microwave power saturation and is a function of the electron spin relaxation times T1 and T2. Fig. 4. Example of a redox titration curve. For each data point, the sample is poised at the potential shown, and the EPR spectrum is taken, if necessary by removing a sample and freezing. The amplitude of some feature in the spectrum is measured and plotted against the potential. The data are fitted to a curve calculated from the Nernst equation, with the maximum signal amplitude and midpoint potential Em as variables.

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