ELECTRON SPIN RESONANCE SPECTROSCOPY | Biological Applications

ELECTRON SPIN RESONANCE SPECTROSCOPY | Biological Applications

344 ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications Biological Applications A F Vanin, Semenov Institute of Chemical Physics, Moscow, ...

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344 ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications

Biological Applications A F Vanin, Semenov Institute of Chemical Physics, Moscow, Russia E E H van Faassen, Universiteit Utrecht, Utrecht, The Netherlands & 2005, Published by Elsevier Ltd.

Introduction The application of electron spin resonance (ESR) to biological studies started from the realization that free radicals play a very important role in enzymatic protein catalysis. In the 1950s Barry Commoner in the USA and Lev Blumenfeld in the USSR were the first to begin testing this hypothesis using ESR spectroscopy on tissue and cell preparations. Subsequent studies confirmed the crucial role of paramagnetic metal complexes for the enzymatic activity of biological materials. Two examples are afforded by the process of mitochondrial respiration and the kinetics of the photosynthetic electron transport chain in plants and bacteria. The paramagnetic centers involve flavin and semiquinone radicals, or short-lived cationic or anionic radical intermediates. In addition, Helmut Beinert in the USA discovered in the late 1950s the presence of stable paramagnetic iron–sulfur centers in frozen animal tissues and cell components. Geometrical information on tertiary protein structure can be obtained if ESR spectroscopy is used to measure the distances between two paramagnetic sites in a protein. Such sites may be native (iron–sulfur clusters or metal ions) or artificially incorporated by means of a paramagnetic label in a well-defined location of the protein. Driven by technological advances, the necessary labeling techniques and distance measurements using pulsed ESR and pulsed electron nuclear double resonance (ENDOR) have undergone very rapid progress in recent years. In recent years, much interest has been focused on the detection of reactive oxygen species (ROS) and nitric oxide (NO) radicals in viable cell cultures and tissues. The spin trapping technique has proved to be very valuable, and its general characteristics have been described elsewhere in this encyclopedia. The complex biochemistry involved requires special techniques for spin trapping ROS and NO in biological materials. Such techniques form the content of the final sections. Finally, spin labeling of proteins, ESR oximetry, and the use of spin probes for investigation of the structure of biomembranes are classic examples of biological ESR. These topics have been discussed elsewhere in this encyclopedia.

Structure and Function of Metalloproteins Enzymes can be considered as highly developed and selective catalysts that control much of the chemistry in living organisms. In nearly all cases, the crucial reaction steps involve a redox reaction mediated by a transition metal ion, for example at the heart of the enzyme. Such electron transfers affect the electronic configuration of the metal ion and the magnetic moment of the ion. Many, though not all, important enzymes have at least one paramagnetic charge state and may be observed in principle using ESR. The ESR spectrum of transition metal ions is remarkably sensitive to the so-called crystal field interactions with the atomic neighbors of the ion (spin–lattice interaction). Their outer electronic states consist of so called d-states, i.e., the five possible states characterized by an orbital angular momentum quantum number L ¼ 2. In an isolated atom, all five orbitals would have the same energy. This degeneracy is partially or completely lifted by the interaction with surrounding ligands and leads to distinct positions of the energy levels. In this way, tetrahedral, octahedral, tetragonal, or square planar surroundings may be easily distinguished. ESR spectra are particularly sensitive to violations of the structural symmetry, such as small distortions of the tetrahedral pyramid or loss of axial symmetry of the metal center. In addition, the ESR spectra may show hyperfine couplings between the electronic spins and the magnetic moments of nearby magnetic nuclei like nitrogen (I ¼ 1), hydrogen (I ¼ 1/2), or many transition metal ions like Cu (I ¼ 3/2), Co (I ¼ 7/2), or Mn (I ¼ 5/2). Additional structural information may be obtained by isotopic substitution of natural nonmagnetic nuclei by magnetic isotopes like 57Fe (I ¼ 1/2, natural abundance 2.2%) or 67Zn (I ¼ 5/2, natural abundance 4%). A table of the most relevant paramagnetic metal ions may be found elsewhere in the encyclopedia.

Distance Measurements in Biomolecules In noncrystalline samples, ESR provides a convenient technique for estimating the distance between paramagnetic sites on a macromolecule. The spatial distance between the sites determines the strength of the spin–spin interaction, which may be experimentally determined using ESR. Such interactions may be

ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications 345

studied in a liquid or frozen solution, glasses, in porous matrices, and in amorphous or crystalline solids. At close distances, the spin–spin interactions are sufficiently strong to affect the shape of continuous wave (CW) ESR spectra. At larger separations these interactions are weak and better resolved using modern pulsed ESR spectroscopy methods like electron spin echo modulation (ESEEM) or pulsed electron– electron double resonance (ELDOR or PELDOR). ESR is very selective as it provides information on only those few sites that carry an electronic magnetic moment. It has proved to be very valuable for macromolecules that cannot be crystallized or that are too large for nuclear magnetic resonance investigations in solution. Although pulsed ESR requires cooling to low temperatures, it can be applied to fully functional proteins embedded in their natural matrix like a lipid cell membrane or attached to a coenzyme. It provides information on protein tertiary structure, conformational changes during enzymatic activity, and the dynamics of protein folding. The paramagnetic sites may be endogenous flavin or semiquinone radicals, cation or anion radical intermediates, or paramagnetic metal centers. Additionally, exogenous spins may be included by site-directed spin labeling. The 3-(methanesulfonylthiomethyl)2,2,5,5-tetramethylpyrrolidin-1-yloxy spin label (also called methane–thiosulfonate) is a nitroxide that has proved to be particularly useful for selective labeling of cysteine residues. It attaches to the protein backbone via a rigid disulfide bridge, which makes the orientation and mobility of the label reflect those of the backbone itself. The spin–spin interactions are caused by either dipolar magnetic coupling between the spins or electronic exchange interaction due to orbital overlap between the two unpaired electrons involved. Such electronic exchange may be direct or mediated through hydrogen bonds or bridging ligands between paramagnetic metal centers. In continuous wave ESR, the coupling strength is best determined via the intensity of the so-called half-field transition, which occurs near half the magnetic field for the usual single quantum transitions. This double quantum transition becomes weakly allowed due to the anisotropic part of the spin–spin interaction (dipolar interaction and anisotropic exchange). The practical experimental range extends to distances up to B12 A˚ for continuous wave ESR and up to 30 A˚ for pulsed excitation of this double quantum coherence (DQC). Far longer distances are accessible by specialized pulsed ESR methods. Particularly useful are double frequency resonance techniques where two different spin transitions are successively excited by pulse sequences with two microwave frequencies. The

Table 1 Selection from ESR methods to determine the distance between paramagnetic sites. The acronyms are explained in the text Technique

Range ˚) (A

Spectral simulation CW 4–20 Intensity of half-field transition CW 4–12 Intensity of half-field transition Pulsed 12–25 DQC Pulsed 20–30 DEER Pulsed 15–70 PELDOR Pulsed 15–130

No. of frequencies used 1 1 1 1 2 2

coherent excitation of two different microwave transitions provides a high selectivity that allows the determination of even very weak coupling between distant spins. The basic PELDOR experiment is based on a three-pulse sequence but suffers from experimental limitations like ‘blind spots’ and ‘dead time’. These problems may be reduced in the more sophisticated four-pulse double electron–electron resonance (DEER) spectroscopy. The most commonly used ESR methods for distance measurement are listed in Table 1.

ESR-Detectable Endogenous Paramagnetic Centers in Animal Tissues, Cells, and Bacteria The majority of endogenous paramagnetic centers detected in animal cells and tissues arise in electron transport chains (ETCs) of mitochondria. They comprise heme or nonheme iron-containing proteins (cytochromes and iron–sulfur proteins, respectively), Cu2 þ -containing proteins, protein-based radical centers (flavine mononucleotide or flavine adenine nucleotides) as well as certain radical intermediates of low molecular weight (ubiquinones). Due to intensive spin–lattice relaxation, the ESR spectra of iron-containing protein components of ETC can be recorded only at cryogenic temperatures in the range 4–80 K. Microwave power saturation of the ESR signals should be avoided at such low temperatures. Figure 1 shows the complex ESR spectra from isolated cardiac mitochondria. They appear as a superposition of spectra from various paramagnetic components of the mitochondrial ETC. They are mainly iron–sulfur centers, denoted as N1, N2, N3 þ 4 (located in complex I, NADH–ubiquinone oxidoreductase), S1 (complex II, succinate–ubiquinone oxidoreductase), and the Rieske iron–sulfur protein (complex III, ubihydroquinone–cytochrome C oxidoreductase). The positions of the components

346 ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications

204 202 200 199

22 217

CYTc OXIDASE S3 22

1.92 1.89 1.86 1.81 1.78

(A) (A)

(B) (B)

(C)

(D)

(C)

(E)

178

194 192 180 186

210 208 208 202

2.10 2.08 2.05 1.99

Center N1 Center N2 N3 Center Center N4 Fe−S of Complex III Figure 1 EPR spectra of electron transport particles (ETPs) from heart mitochondria from titration with NADH. (A) ETPs treated as other samples except that no reductant was added; (B)–(E) reduced with increased dose of NADH. (Reproduced with permission from Orme-Johnson N et al. (1974) Electron paramagnetic resonance-detectable electron acceptors in beef heart mitochondria. Journal of Biological Chemistry 249: 1922–1939.)

(i.e., the g-tensor values) are shown in Figures 1 and 3. Similar spectra are characteristic of various isolated animal tissues. As shown in Figures 2 and 3, the shape of the spectrum depends on the redox state of the paramagnetic centers. Full reduction of the mitochondrial ETC components is obtained upon depletion of oxygen in tissue. In the oxidized state, the ESR spectra appear as superpositions from cytochromes, the copper center in cytochrome oxidase, and a high-potential iron–sulfur center, S3 (complex II). An additional signal arises from the iron–sulfur

Figure 2 EPR spectra of whole pigeon heart at different reductive states of the electron carriers (the extent of reduction increases from A to C). (Reproduced with permission from OrmeJohnson N et al. (1974) Electron paramagnetic resonancedetectable electron acceptors in beef heart mitochondria. Journal of Biological Chemistry 249: 1922–1939.)

cluster of mitochondrial aconitase, the enzyme from the Krebs cycle. The ESR spectra also give valuable information on pathologies like cancer. Figure 4 shows that the iron–sulfur signals from hepatoma mitochondria in Morris rats are correlated with the mitochondrial respiratory activity. In the slow-growing hepatoma16 tumors, the iron–sulfur signals from reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase are smaller than from surrounding nonaffected liver tissue or liver from healthy rats. In the rapidly growing hepatoma-7777 cells, the ESR signals of iron–sulfur centers are even more diminished. The data show that the abnormal metabolic characteristics of some tumors are related to specific defects in the iron–sulfur components of the ETC. Recent ESR data suggest that the final step of proton translocation in complex I involves an endoenergetic electronic disproportionation in the ubisemiquinone pair. If tightly coupled and functioning bovine heart submitochondrial particles are snap

ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications 347

2.08 2.02 g = 2.1 2.05

1.92

2.08 2.02

1.92 2.0 1.94

1.89

2.41

1.94 1.89

2.05 2.0

2.25

1.86

1.86

(D)

(B)

(C)

(A)

S−3 +2

S− 3

Cu

+2

Cu

N−1b N− 2 S −1 N−3 + 4

N−1b N− 2 S−1 N− 3 + 4

Figure 3 EPR spectra of isolated tissues of mouse heart (A,B) and liver (C,D). (A,B) The tissues after isolation from the organism; (B,D) after low temperature oxidation (by maintaining isolated tissues at  121C for 5 h). (Reproduced with permission from Burbaev D et al. (1975) ESR spectra of animal tissues in vitro. Biofyzika (Russian) 20: 1062–1067.)

58K

2.03 1.94

host liver tumor (16)

host liver tumor (7777)

30 K

211 ×2

1.92 17K

194 190

2.03 2.09 2.06

13 K

206 209 ×1

2.01

201

189

1.89 193 31 1.93

33 Kgouss

35

Figure 4 EPR spectra of reduced iron–sulfur centers in mitochondria prepared from slow growing Morris hepatoma-16 (left panel) or rapidly growing Morris hepatoma-7777 and host liver (right panel), measured at 58 and 17 K, or 30 and 13 K, respectively. Mitochondrial suspensions were brought to anaerobiosis by incubating with glutamate þ malate for 10 min. (Reproduced with permission from Ohnishi T et al. (1973) Electron paramagnetic resonance studies of iron-sulfur centers in mitochondria prepared from three Morris hepatomas with different growth rates. Biochemistry and Biophysics Research Communications 55: 372–381.)

frozen in the presence of an NADH substrate, strong ESR signals at g ¼ 2.00 show that one ubisemiquinone of this pair is in a radical state (spectra shown in Figure 5). It undergoes fast spin–spin relaxation

due to interaction with the iron–sulfur center, N2, located at a distance of only 10 A˚. This ubisemiquinone signal was not detected in uncoupled submitochondrial particles. The data suggest that energy

348 ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications 2.025 2.05 2.10

g = 2.42

2.25

2.03 1.97 1.94

1.91

2.00

1.93 (N2.g )

(N2, g )

(A) 1.86 1.88

(A)

H

2.025

g = 2.00 g =1.98 g = 2.1 g = 2.07 g = 2.035

2.00

(B)

(B) 300

320

340

360

380

Magnetic field (mT) Figure 5 EPR spectra of uncoupled (A) and tightly coupled (B) submitochondrial particles during steady-state NADH oxidation. Recordings were made with microwave power 2 mW and temperature 16 K. (Reproduced with permission from Vinogradov A et al. (1995) Energy-dependent Complex I-associated ubisemiquinones in submitochondrial particles. FEBS Letters 370: 83–87.)

transduction in mitochondria requires the participation of bound ubisemiquinones as well as the iron– sulfur center N2. Analysis of these mitochondrial ESR spectra sheds light on the nature, redox state, and quantity of the ETC components. This has been very helpful in understanding the redox behavior of enzymatic centers and in deducing their orientation in mitochondrial membranes. It should be kept in mind that the ESR spectra reflect the ETC components in the frozen state. As such, the spin and redox states of the paramagnetic centers may differ from those of the active enzyme at a physiological temperature. Oxidized cytochrome, P450, is the terminal component of the microsomal ETC. Its g-values at g ¼ 2.42, 2.25, and 1.91 are easily distinguishable from those of components in the mitochondrial ETC. Spectra from this enzyme are often detected in

Figure 6 ESR spectra of liver tissue from mice at 77 K: (A) control animal on normal diet; (B) mouse on drinking water with 0.3% nitrite for 7 days. The nitrite consumption induces formation of dinitrosyl-iron (DNIC 0.03) and nitrosyl-heme complexes, at g ¼ 1.98. (Reproduced with permission from Varich V (1979) Changes in amounts of dinitrosyl non-heme iron complexes in animal tissues depending on animal growth. Biofyzika (Russian) 24: 344–347.)

isolated liver and kidney tissue at temperatures below 100 K (Figure 6). The redox processes in the microsomal ETC ensure detoxication of many xenobiotics as well as the biosynthesis and biodegradation of endogenous compounds like steroids or fatty acids. The detoxication defense of many bacteria, insects, fishes, yeasts, and plants involves a wide range of cytochromes. Like cytochrome P450, these cytochromes have paramagnetic charge states. ESR spectroscopy has proved to be very useful for studying the catalytic cycle of this class of enzymes. A particular success was the detection of the socalled Compound 1, a highly redox-active intermediate porphyrin radical–cation–Fe(IV)QO complex. Paramagnetic centers are found in many specific enzymes and proteins that function in animal tissues and bacteria (oxygenases, sulfite or nitrite reductases, xanthine oxidase, nitrogenase, etc.) besides mitochondria and microsomes. The catalytic site can include Mo, Cu, Co, Ni, Mn, and other metal ions. However heme– and iron–sulfur centers constitute the majority of the paramagnetic centers found outside the mitochondria.

ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications 349

In recent years, the interest in the physiological role of nitric oxide radicals has greatly stimulated the investigation of iron–nitrosyl complexes. The generation of nitric oxide in mammalian tissues, cultured cells, or bacteria is usually accompanied by the formation of paramagnetic mononitrosyl–heme iron complexes and dinitrosyl–nonheme iron complexes (DNICs). The respective ESR signals of the complexes are shown in Figure 6. Nitric oxide is recognized now as a signaling and regulatory radical that influences diverse physiological and biochemical processes. In biosystems, it is generated either enzymatically by NO synthases or nonenzymatically (mainly from nitrite). A possible physiological role of the paramagnetic nitrosyl–iron complexes is now being hotly debated and investigated. Various forms of DNIC may be distinguished according to the structure of the thiolate group that ligates to the central iron atom. The preliminary results suggest that DNICs may be implicated in the redox chemistry of S-nitrosothiols. The latter are known to have antioxidant as well as signaling functions in cells and tissues. Recently DNICs have been found in plant leaves upon suppletion of NO from an exogenous source or generated endogenously from nitrite. It is reasonable to suggest that the formation of DNICs from nonheme iron prevents iron precipitation in the form of hydroxide complexes. This would increase the iron availability for various intracellular components, particularly for chloroplasts.

ESR-Detectable Endogenous Paramagnetic Centers in Photosystems of Plants ESR spectroscopy has been very useful in studying the photosynthetic function in plants and bacterial photosystems. It had been known for a long time that illumination causes free radical ESR signals in photosynthetic systems, and subsequently the importance of nonheme iron and then manganese complexes was discovered. The photosynthetic activity of plants is based on two supramolecular assemblies, photosystems I and II, which act in tandem. Each photosystem consists of a light harvesting antenna structure containing chlorophyll dye molecules, and a cascade of electron acceptors that act in succession as an electron transport chain for charge separation. The ESR signals from the photosystems may be observed at cryogenic temperatures and depend sensitively on the state of illumination. Figure 7 shows the spectra of photosystem I from spinach chloroplasts. Under steady illumination, they appear as a superposition of an intense g ¼ 2.00 free radical line

Dark

Light

g = 2.05 3100

g = 1.94

3200 3300 3400 Magnetic field (G)

g = 1.86

3500

Figure 7 Photoreduction of a bound Fe–S center in intact spinach chloroplasts after illumination at 77 K. Recordings were made at 10 K. (Reproduced with permission from Malkin R and Bearden A (1971) Primary reactions of photosystem: photoreduction of a bound ferredoxin at low temperature as detected by ESR spectroscopy. Proceedings of the National Academy of Sciences of the USA 68: 16–19.)

from oxidized pigment P700 þ and an anisotropic ESR signal at g-values of 2.05, 1.94, and 1.86 assigned to a reduced Fe–S center. A quantitative comparison of the P700 þ content and paramagnetic Fe–S center, as well as a comparison of their kinetics characteristics at temperatures 10–100 K, has shown that photosystem I has a good correlation between P700 oxidation and the ESR intensity from the Fe–S center. This center turned out to be the secondary electron acceptor from the electron transport chain. The nature of the primary quinone–iron acceptor is revealed using ESR if the electronic pathway to the secondary acceptor is blocked. In the reduced state, this so-called X center has a broad spectrum with g ¼ 1.78, 1.88, and 2.08. Photoinduced oxidation of water into dioxygen is the central step in photosynthesis. It is carried out through a characteristic tetranuclear manganese cluster in photosystem II. This cluster is the active site for water oxidation by formation of the so-called oxygen-evolving complex (OEC). Photosynthetic oxidation of water is a highly complex mechanism involving a sequence of five intermediate states, Si. It has been proved that the first intermediates, S0–S3, of the Mn–OEC donor complex are sufficiently stable to be detected in frozen samples using ESR. For example, Figure 8 shows the spectrum of the S2 state. The signal is mainly accepted to arise from a S ¼ 1/2

350 ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications

Control

(B) (A) g=4

g =2

Fluoride 0.332

0.333

0.334

0.335

0.336

0.337

0.338

Field ( T )

900

4100 Field (G)

Figure 8 EPR spectra associated with the oxygen-evolving center in photosystem II preparations: the multiline EPR signal and the signal at g ¼ 4.1 (top panel) from the preparation in the S2 state (illumination at 195 K); EPR signal (shown at the bottom) produced by continuous illumination of a sample at 200 K in the presence of 20 mmol l  1, 20 mM fluoride. Recordings were made at 10 K. A narrow ESR line from a spurious free radical near g ¼ 2.0 was removed from the spectra. (Reproduced with permission from Yachandra V, Sauer K, and Klein M (1996) Manganese cluster in photosynthesis: Where plants oxidize water to dioxygen. Chemical Reviews 96: 2927–2950.)

antiferromagnetically exchange-coupled Mn4(III, IV3) cluster. A multiline structure is located near g ¼ 2.00 and shows well-resolved hyperfine couplings from the manganese nuclei. The additional low-field signal at g ¼ 4.1 is suggested to arise from some clusters existing in a high-spin S ¼ 3/2 or S ¼ 5/2 electron configuration. The g ¼ 4.1 signal can also be formed through infrared illumination of the S ¼ 1/2 state at 65 K. It is also observed in photosystem II upon exposure to F  anions. Such samples lose the multiline structure near g ¼ 2.00 as well as the capacity to generate O2. Interestingly, the halide anion Cl  is an essential cofactor for O2 production through photosystem II. The anion may be replaced by Br  with full retention of activity. Moreover, either Cl  or Br  is required for the multiline structure near g ¼ 2.00, which is characteristic of the S2 state. It strongly suggests that the halide anion is a ligand of the Mn atoms in the cluster, but up to now attempts to detect

Figure 9 ESR spectra of free thyrosyl radical TyrD photogenerated in Mn-depleted photosystem II at 4 K (A) and warming to 200 K (B). The spectral change near 0.3362 T is attributed to a thermally activated deprotonation of the YDþ radical. (Reproduced with permission from Faller P, Goussias C, Rutherford AW, and Un S (2003) Resolving intermediates in biological proton-coupled electron transfer: A tyrosyl radical prior to proton movement. Proceedings of the National Academy of Sciences of the USA 100: 8732–8735.)

the hyperfine interaction from the halide ion have failed. The Mn cluster is not directly photoactivated itself but via an indirect redox process involving a tyrosine intermediate. Upon photoactivation of the primary donor in photosystem II, the excited electron is injected into the electron transport chain and removed. The oxidized primary donor, P680 þ , receives an electron from a nearby tyrosine YZ. The resulting Yþ Z radical intermediate oxidizes the tetranuclear Mn–OEC to a high valence state that is capable of oxidizing water. This short-lived Yþ Z radical intermediate has ms lifetime and has been studied with pulsed ESR and optical spectroscopy. In addition, illuminated photosystem II contains a long lived Yþ D tyrosyl radical, which is stable at cryogenic temperatures. The ESR spectra reveal a thermal relaxation of the molecular structure around this Yþ D radical at 80 K (see Figure 9). The spectral changes were attributed to a thermally induced deprotonation of the Yþ D cation.

Spin Trapping of Nitric Oxide NO radicals are paramagnetic with a degenerate ground electronic state, which precludes direct ESR detection except at cryogenic temperatures in the

ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications 351

frozen state. When bound with various protein amino acid residues, it forms paramagnetic centers that may be observed even at room temperature. The ESR spectra resemble those from stable nitroxyl radicals like 2,2,6,6-tetramethyl piperidino oxy nitroxide (TEMPO). As these centers are susceptible to oxidation by superoxide, their concentrations usually remain below the detection threshold in biosystems. Spin trapping of NO molecules provides a good alternative. Various derivatives of dithiocarbamate ligands are known to enhance considerably the affinity of ferrous ions for NO molecules. The straightforward binding of NO to the Fe(II)–(dithiocarbamate)2 complex is often referred to as a trapping reaction of NO. The adduct is a paramagnetic mononitrosyliron complex (MNIC) with dithiocarbamate ligands that can be detected and evaluated using ESR spectroscopy even at ambient temperature. Figure 10 shows the spectra of MNICs in the frozen and liquid solution states, respectively. The triplet structure is caused by hyperfine interaction of the unpaired electron with the nitrogen nucleus (I ¼ 1) of the NO ligand. The moderate magnitude of this hyperfine splitting demonstrates that the unpaired electron is largely transferred from the NO to the iron atom, which must have a formal monovalent

g = 2.035

g = 2.03

2.02

(B) (A) 1.2 mT B 1.2 mT B

(D)

(C)

Figure 10 EPR spectra of MNIC–MGD complexes in aqueous solution, including 56Fe (A and B) or 57Fe (C and D). Recordings were made at 77 K (A and C) or ambient temperature (B and D). Isotopic substitution of 56Fe (I ¼ 0) with 57Fe (I ¼ 1/2) results in changes in the signal shape that are due to the manifestation of an additional HFS induced by interaction of the unpaired electron with the 57Fe nucleus (I ¼ 1/2). EPR spectra from MNIC–DETC. (Reproduced with permission from Mikoyan et al. (1997) Complexes of Fe2 þ with diethyldithiocarbamate or N-methyl-D-glucamine dithiocarbamate as traps of nitric oxide in animal tissues: Comparative investigations. Biochimica Biophysica Acta 1336: 225–234.)

redox state of Fe(I). Depending on the hydrophobicity of the dithiocarbamate ligands, the MNIC complexes localize preferably in either the hydrophobic or the hydrophilic compartments of cells of tissues. Widely used dithiocarbamates include diethyldithiocarbamate (DETC) and N-methyl-D-glucamine dithiocarbamate (MGD), which give rise to hydrophobic and hydrophilic MNICs, respectively. After suppletion of free iron and dithiocarbamate ligands, such MNICs are detectable in many biosystems using ESR spectroscopy, from cultured viable mammalian cells and tissues to living plants (Figure 11). The NO trapping and MNIC formation proceeds in vivo, whereas the actual ESR detection and quantification of MNICs is usually carried out ex vivo. Recent advances in sensitivity have even allowed MNIC detection in small living mammals. In combination with ESR imaging, MNIC even allows NO tomography revealing the distribution of MNICs in the body of a small animal. ESR spectroscopy has provided the first unequivocal proof that NO synthesis from L-arginine using NOS enzymes is the only relevant source of NO in mammals: MNIC formation can be prevented by infusion of NOS inhibitors. In addition, isotopic labeling of L-arginine with 15N (I ¼ 1/2) isotopes leads to the formation of MNICs with a doublet structure instead of the usual nitroxide triplets obtained with the natural 14N isotope (Figure 11). In this context, it should be mentioned that modest quantities of NO may also be released from nitrite via nonenzymatic pathways under conditions of acidosis. It is noteworthy that Fe–dithiocarbamate traps are normally used in the millimolar range. As such, MNIC formation will provide the dominant reaction pathway for endogenous NO molecules under normal physiological conditions. In particular, we can neglect the alterative pathway involving superoxide anions that is present in living systems at a concentration of not more than a few micromoles. Nevertheless, as will be discussed below, the presence of superoxide has a big effect on the MNIC lifetime. Experimental MNIC spectra obtained in tissues usually have to be corrected for the presence of an additional broad background caused by paramagnetic Cu(II)–DETC complexes. DETC is a sufficiently good chelator to scavenge spurious free copper and even extract Cu(II) ions from certain endogenous enzymes like superoxide dismutase (SOD). The interpretation of NO trapping experiments using Fe–dithiocarbamate complexes is particularly complicated by the redox activity of both Fe–dithiocarbamate complexes and their nitrosyl adducts. The Fe(II)–dithiocarbamate complexes are easily

352 ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications

g = 2.035 2.02

2.035 1.98 2.07 2.02 g = 2.035 2.02

A B C D

g = 2.035 2.02

A B C D

A

(A)

30 (B)

10

(A)

(A)

(B)

(B)

(C)

(C)

(D)

(D)

(E)

(E)

B

C

3 mT B

4 mT B (C)

1 (D)

15

(F) 2.6 mT mT 320

330

B

340

Figure 11 Left panel: EPR spectra of MNIC–DETC complexes from a mouse liver preparation from control animal injected with DETC (A), an animal injected with bacterial lipopolysaccharide (LPS) (4 h) þ DETC (B), an animal injected with LPS (4 h) þ DETC and Fe–citrate complex (C). (D) EPR spectra of a mouse spleen preparation from an animal injected with LPS (4 h) þ DETC and Fe–citrate complex. The MNIC–DETC complex gives an EP signal at g> ¼ 2.035, g8 ¼ 2.02 and triplet HFS at g>. A,B,C, and D indicate the quartet HFS of the EPR signal due to the Cu2 þ –DETC complex formed in the liver. The EPR signal with the components at g ¼ 2.07 and 1.98 in (D) is due to a nitrosyl–heme iron complex. Recordings were made at 77 K. Middle panel: The shapes of the EPR signal of MNIC–DETC including 14NO (A) and 15NO (B) only, or at various ratios of these ligands: 15NO: 14NO ¼ 3:7 (C), 1:1 (D), 2:1 (E), 4:1 (F). Recordings were made at 77 K. Right panel: EPR spectra of macrophages (107 cells per sample) stimulated by LPS for 0 (A), 5 (B), and 11 h (C–E) and subsequently incubated with DETC, FeSO4, and LPS for 2 h. (15NG L-arginine) was present in (D) and (E) for the last 5 h of incubation. Spectra were recorded at 77 K. (A–C) indicate the position of three low field HFS components from Cu2 þ (DETC)2 complexes. g ¼ 2.035 and 2.02 indicate the position of g> and g8 of the NOFe2 þ (DETC)2 complex. (Reproduced with permission from Kubrina L et al. (1992) EPR evidence for nitric oxide production from guanidino nitrogen of L-arginine in animal tissues in vivo. Biochimica Biophysica Acta 1099: 223–237 and Vanin A et al. (1993) The relationship between L-arginine-dependent nitric oxide synthesis, nitrite release, and dinitrosyl-iron complex formation by activated macrophages. Biochimica Biophysica Acta 1177: 37–42.)

Table 2 Spectroscopic properties of iron(II) and iron(III)–MGD complexes in aqueous solutionsa Iron–carbamate complex

Absorption wavelength (nm)

Extinction coefficient (l mol  1)

Color

Fe(II)–MGD Fe(III)–MGD NO–Fe(II)–MGD NO–Fe(III)–MGD

– 340, 385, 520 314, 368, 450 No peaks

– 20 000, 15 000, 3 000 18 000, 12 500, 5 600 B20 000 at 350 nm

Clear, diamagnetic Orange-brown, paramagnetic (S ¼ 3/2) Deep green, paramagnetic (S ¼ 1/2) Yellow, diamagnetic

a Data adapted with permission from Vanin A et al. (2000) Redox properties of iron–dithiocarbamates and their nitrosyl derivatives: Implications for their use as traps of nitric oxide in biological systems. Biochimica Biophysica Acta 1474: 365–377.

oxidized to Fe(III)–dithiocarbamate complexes by dissolved oxygen. In an aqueous solution, the rate constant of the reaction for Fe(II)–MGD complexes was determined as 5  105 l mol  1 s  1 at ambient temperature. Superoxide radicals react considerably faster with a rate of 3  107 l mol  1 s  1. The Fe(III)– (dithiocarbamate)3 complexes can also bind NO molecules, which results in the formation of diamagnetic iron(III) nitrosyl derivatives. However, the latter are rather unstable and slowly transform into paramagnetic MNIC–dithiocarbamate under anaerobic

conditions. This transformation may be accelerated by addition of reducing agents like ascorbate. Interestingly, the presence of the NO ligand seems to facilitate this reduction step as the reduction of the iron from the Iron(III) to the Iron(II) state is considerably slower in the absence of NO. The redox conversion may be followed spectroscopically using ESR as well as optical absorption as the redox state and the presence of the NO ligand strongly affect the color of the iron complex. Table 2 lists the optical and ESR properties.

ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications 353

We stress that ESR spectroscopy only detects the paramagnetic versions of MNIC. As such, the ESR intensity may only reflect the NO levels in tissues or biomaterials if all the iron is reduced to the Iron (II) state. Three pathways for this transformation are known in biomaterials. The first is straightforward reduction of diamagnetic MNICs by endogenous reducing agents like glutathione and ascorbate. Second is a mechanism of reductive nitrosylation similar to that proposed for nitrosyl ferrihemoproteins, where the NO ligand reduces the iron to the ferrous state, followed by release of NO þ from the complex. Nevertheless, it should be kept in mind that these mechanisms do not ensure complete reduction of all complexes to the iron(II) state. An additional problem concerns the stability of paramagnetic MNICs in biomaterials. It has recently been discovered that paramagnetic MNICs react rapidly with superoxide and peroxynitrite. The reaction products are ESR-silent nitrosocomplexes. In other words, the adducts are formed but do not survive. This complication can be partially redressed by the so-called ABC method. Its basic idea is to overwhelm these alternative pathways by bolus injection of a known quantity of exogenous MNIC to a cultured cells or animal tissue. It acts as a very efficient scavenger of superoxide anions and peroxynitrite, so that endogenously formed MNIC adducts survive. This experiment is repeated in the presence of a NO synthase inhibitor. The endogenous NO levels are estimated from the difference between these two experiments: first experiment ¼ A þ B  C, second experiment ¼ A  C, where A is the signal from the exogenous MNIC bolus injection and B is the signal due to endogenous NO production. The quantity C accounts for the endogenous MNIC adducts transformed into the ESR-silent state by superoxide and/or peroxynitrite. Figure 12 illustrates the advantage of the ABC method over the ‘conventional’ method in cultured endothelial BEND3 cells. The ABC method allows detection of a larger fraction of endogenously produced NO. As such, its gives a better representation of NO levels in biomaterials, tissues, and living systems. Still, we cannot exclude the possibility that an unknown fraction of NO ends up in ESR-silent nitroso compounds. Organic spin traps are also used for NO detection in chemical and biological systems. For example, 2,5dimethylhexadiene, when introduced into degassed organic solutions of NO, yields a three-line ESR signal, tentatively assigned to 2,2,5,5-tetramethyl-1pyrrolnoxyl. Paramagnetic nitronyl nitroxides change their ESR spectrum when they react with NO to form a paramagnetic imino nitroxide adduct.

g = 2.035 (A)

1

(B)

1 2.5

(C) (D)

2.5 mT

330

334

338

342

346

Figure 12 EPR spectra of MNIC–DETC complexes formed in suspensions of viable endothelial cells. (A) In the absence of NOS inhibitor, N-nitro-L-arginine (NLA); Endogenous and exogenous MNIC–DETC make contributions to the signal. (B) With 1 mmol l  1 NLA. Only exogenous MNIC–DETC makes a contribution to the signal. (C) Computed difference (A–B), showing the endogenous MNIC–DETC formed NO synthesis. (D) Reference spectrum showing MNIC–DETC complexes formed endogenously by conventional NO trapping. Spectra were recorded at 77 K. The relative gain settings are shown at the right side of the spectra ABC method. (Reproduced with permission from Vanin A et al. (2001) Antioxidant capacity of mononitrosyl-iron-dithiocarbamate complexes: Implications for NO trapping. Free Radical Biology and Medicine 30: 813–824.)

For example, PTIO or carboxy-(2-(p-carboxy)phenyl4,4,5,5-tetramethylimidazoline 3-oxide-1-oxyl) reacts efficiently with NO to form paramagnetic carboxy(2-(p-carboxy)phenyl-4,4,5,5-tetramethylimidazoline 3-oxide). However, in viable cells and tissues, such adducts are easily transformed into diamagnetic hydroxylamines. This property severely limits the ESR application of these traps in bioassays. Potentially valuable information may be obtained from the class of fluorescent cheletropic traps. These have weak red fluorescence and react with NO to form nonfluorescent paramagnetic adducts. The latter are rapidly transformed into a diamagnetic hydroxylamine with strong blue fluorescence, which is easily observable using fluorescence microscopes and spectrometers. Nevertheless, iron–dithiocarbamate complexes form the basis of the most valuable and reliable ESR technique for detecting NO in living tissues.

Spin Trapping of Oxygen Radicals Singlet oxygen, triplet oxygen, ozone, superoxide anions and the hydroxyl radicals are all fairly reactive oxygen species. Singlet oxygen is diamagnetic and unobservable using ESR, whereas the remaining

354 ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications

species are paramagnetic. They cannot be observed directly using ESR due to line broadening or the short lifetimes of the species. In biological materials, ozone does not occur in significant quantities and will not be considered further. Superoxide and hydroxyl radicals have been recognized as major and inseparable agents in the development of physiological pathologies. In biological research, these radicals always appear in the company of the nonradical hydrogen peroxide, to which they are linked via a complex series of chemical reactions (dismutation and iron-mediated Fenton chemistry are discussed elsewhere). Evolution has developed elaborate enzymatic defenses to reduce the in vivo levels of superoxide and hydrogen peroxide, in the form of superoxide dismutase and catalase, respectively. Nevertheless, minute quantities of oxygen radicals may escape the defenses and contribute to undesirable oxidative stress and oxygen toxicity. The superoxide anion O2 is modestly reactive when compared with most other radicals but still is a stronger one-electron reductant and oxidant than O2 itself. This S ¼ 1/2 radical can be observed using ESR only in exceptional cases where the spin relaxation is reduced sufficiently. In biological systems, its concentration remains too low and the linewidth too broad for observation. In the late 1960s, nitroso and nitrone compounds were introduced as spin trapping agents to detect superoxide radicals. The traps are diamagnetic, but their reaction with the primary radical leads to the formation of a more stable paramagnetic reaction product in the form of a nitroxide (adduct in spin trapping terminology):

dimerize, leaving only a small concentration of the monomer, which alone is capable of the trapping reaction. Some traps like MNP are sufficiently volatile to be lost from solutions through degassing via nitrogen bubbling. Wider applications include nitrone spin traps, which are light insensitive, nontoxic, and lead to reasonably robust adducts. The adduct spectra do not distinguish between different primary radicals as these get inserted at the b-position further away from the nitroxide center. Phenyl-tert-butylnitrone (PBN) and 5,5-dimethylpyrroline (DMPO) are most widely used for superoxide detection in hydrophobic and hydrophilic compartments of biological samples, respectively. DMPO is the standard of choice for application in cell cultures as it readily crosses cell membranes. The spectrum of its superoxide adduct, DMPO–OOH, is shown in Figure 13. The interpretation of the trapping experiments is complicated by the poor stability of DMPO–OOH,

H3C

H

H

H

H

H3C

H OOH

N

10 G

O

paramagnetic primary radical þ diamagnetic trapparamagnetic adduct

The ideal spin trap should be stable, with rigorously stable adducts. The adduct spectra should depend distinctively on the primary radical to allow unambiguous identification of the latter. In reality, the adducts are susceptible to hydroxylation into a diamagnetic hydroxylamine that is ESR silent. This hydroxylation is a serious complication for radical detection in biological systems, particularly in blood samples. Compared with nitrones, nitroso compounds like 2-methyl-2-nitrosopropane (MNP) incorporate the superoxide radical closer to the nitroxide center. Therefore, the adduct spectra provide more information on the nature of the primary radical, making its identification easier. But nitroso traps and their adducts are more susceptible to thermal, photochemical, and hydroxylative degradation as well as -ene addition. They have limited solubility and tend to

H3C H3C

H

H

H

H N

H OH

O

Figure 13 Room temperature spectra of DMPO–OOH adduct (top) and DMPO–OH adduct (bottom) in liquid solution.

ELECTRON SPIN RESONANCE SPECTROSCOPY / Biological Applications 355

15 G

blood, the highly reactive 1-hydroxy-3-carboxy2,2,5,5-tetramethylpyrrolidine (CP–H) has been applied, in spite of a serious susceptibility to artificial adduct formation via secondary pathways (spurious metal ions, peroxynitrite, nonoxygen radicals). In contrast to superoxide, the hydroxyl radical is one of the most reactive substances known. It reacts rapidly with nearly every substance at rates limited only by the time needed to approach its reagent diffusively. As such, it may become difficult to detect by spin trapping in biological materials, where many alternative reaction pathways compete with the trapping reaction. DMPO has proved to be useful in biological systems because the stability of the DMPO–OH adduct comfortably exceeds that of the adducts formed with PBN of a-(4-pyridyl 1-oxide)-Ntert-butylnitrone (4-POBN) spin traps.

Figure 14 Room temperature spectra of DEPMPO–OOH adduct (top) and DEPMPO–OH adduct (bottom) in liquid solution.

See also: Electron Spin Resonance Spectroscopy: Principles and Instrumentation; Specialized Techniques. Nitric Oxide.

which spontaneously dismutates into the very same DMPO–OH adduct that results from the trapping of the hydroxyl radical. Usually, additional experiments in the presence of effective superoxide scavengers like SOD are needed before the primary radical can be identified unambiguously. It should be noted that the rates of the trapping reaction depend sensitively on the pH. For DMPO, the reaction rate with protonated superoxide exceeds that of the superoxide anion by nearly three orders of magnitude. The recently developed nitrone 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) leads to adducts with better stability. Additionally, its decomposition does not lead to potentially misleading DEPMPO–OH adducts. This important advantage has a price: the trap cannot penetrate cell membranes, and the ESR spectra appear as the spectral superposition of two isomers in varying concentrations (Figure 14). Usually, spin traps exhibit fairly small reaction rates with superoxide, of the order of 1–20 l mol  1 s  1 at room temperature. Therefore, fairly long incubations times (B1–30 min) and high trap concentrations (10–50 mmol l  1) are required to accumulate sufficient adducts and to capture a substantial fraction of the superoxide in the assay. Recently, traps with higher reaction rates have become available that can be applied at high micromolar concentrations. For

Further Reading Beinert H and Albracht SPJ (1982) Iron–sulfur proteins. New insight and unresolved problems. Biochimica Biophysica Acta Reviews on Bioenergetics 683: 246–277. Berliner LJ (ed.) (1998) Spin labeling: The next millennium. In: Biological Magnetic Resonance, vol. 14. New York: Plenum. Berliner LJ, Eaton SS, and Eaton GR (eds.) (2000) Distance measurements in biological systems by EPR. Biological Magnetic Resonance, vol. 19. New York: Kluwer Academic. Berliner LJ and Swartz HM (eds.) (2000) In vivo EPR: Theory and application. In: Biological Magnetic Resonance, vol. 18. New York: Kluwer Academic. Henry Y, Guissani A, and Ducastel D (1996) Nitric Oxide Research from Chemistry to Biology: EPR Spectroscopy of Nitrosylated Compounds. Austin: Landes Bioscience. Palmer G (2000) Electron paramagnetic resonance of metalloproteins. In: Que L (ed.) Physical Methods in Bioinorganic Chemistry. Sausalito: University Science Books. Rosen GM, Britigan BE, Halpern HJ, and Pou S (1999) Free Radicals: Detection and Spin Trapping. New York: Oxford University Press. Schweiger A and Jeschke G (2001) Principles of Pulse Electron Paramagnetic Resonance. Oxford: Oxford University Press. Vanin A, Huisman A, and van Faassen E (2002) Iron dithiocarbamate as spin trap for nitric oxide: Pitfalls and successes. Methods in Enzymology 359: 27–42.