Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A~200 G, g~2.186, g~2.051). The pulsed EPR data show that one of the ligands to Cu²⁺ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu²⁺ ion is observed by pulsed electron–nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.

The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. I. Light sensitivity and magnetic hyperfine interactions as observed by electron paramagnetic resonance

The hydrogen-activating cluster (H cluster) in [FeFe]-hydrogenases consists of two moieties. The [2Fe]_H subcluster is a (L)(CO)(CN)Fe(μ-RS₂)(μ-CO)Fe(CysS)(CO)(CN) centre. The Cys-bound Fe is called Fe1, the other iron Fe2. The Cys-thiol forms a bridge to a [4Fe–4S] cluster, the [4Fe–4S]_H subcluster. We report that electron paramagnetic resonance (EPR) spectra of the ⁵⁷Fe-enriched enzyme from Desulfovibrio desulfuricans in the H_ox–CO state are consistent with a magnetic hyperfine interaction of the unpaired spin with all six Fe atoms of the H cluster. In contrast to the inactive aerobic enzyme, the active enzyme is easily destroyed by light. The [2Fe]_H subcluster in some enzyme molecules loses CO by photolysis, whereupon other molecules firmly bind the released CO to form the H_ox–CO state giving rise to the so-called axial 2.06 EPR signal. Though not destroyed by light, the H_ox–CO state is affected by it. As demonstrated in the accompanying paper [49] two of the intrinsic COs, both bound to Fe2, can be exchanged by extrinsic ¹³CO during illumination at 2 °C. We found that only one of the three ¹³COs, the one at the extrinsic position, gives an EPR-detectable isotropic superhyperfine interaction of 0.6 mT. At 30 K both the inhibiting extrinsic CO bound to Fe2 and one more CO can be photolysed. EPR spectra of the photolysed products are consistent with a 3d ⁷ system of Fe with the formal oxidation state +1. The damaged enzyme shows a light-sensitive g=5 signal which is ascribed to an S=3/2 form of the [2Fe]_H subcluster. The light sensitivity of the enzyme explains the occurrence of the g=5 signal and the axial 2.06 signal in published EPR spectra of nearly all preparations studied thus far.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO₂. A unique [NiFe₄S₄] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH•Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe₃S₄] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step.     

Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover: evidence for S = 3/2 spin states from reduced MoFe-protein intermediates

Rapid-freezing experiments elicited two transient EPR signals, designated 1b and 1c, during Azotobacter vinelandii nitrogenase turnover at 23 degrees C and pH 7.4. The first of the signals to form, signal 1b, exhibited g values of 4.21 and 3.76. Its formation was at the expense of the starting EPR signal (signal 1a with g values of 4.32, 3.66, and 2.01). The second signal to arise, signal 1c, with a characteristic g value of 4.69, formed very slowly and was always of low intensity. Both signals occurred independently of the substrate being reduced. Increased electron flux through the MoFe protein caused these signals to form more rapidly. Moreover, after a MoFe-protein solution had been pretreated (using conditions of extremely low electron flux) to set up an equimolar mixture of its resting state and one-electron reduced state, these signals appeared even more rapidly when this mixture was exposed to an excess of the Fe protein. We have simulated the kinetics of formation of these EPR features using the published kinetic model for nitrogenase catalysis [Lowe, D. J., and Thorneley, R. N. F. (1984) Biochem. J. 224, 887-909] and propose that they arise from reduced states of the MoFe protein and reflect different conformations of the FeMo cofactor with different protonation states.     

Measurement of oxygen concentrations in the intact beating heart using electron paramagnetic resonance spectroscopy: A technique for measuring oxygen concentrationsin situ

Electron paramagnetic resonance (EPR) spectroscopy can be applied to measure oxygen concentrations in cells and tissues. Oxygen is paramagnetic, and thus it interacts with a free radical label resulting in a broadening of the observed linewidth. Recently we have developed instrumentation in order to enable the performance of EPR spectroscopy and EPR oximetry in the intact beating heart. This spectrometer consists of 1–2-GHz microwave bridge with the source locked to the resonant frequency of a specially designed lumped circuit resonator. This technique is applied to measure the kinetics of the uptake and clearance of different free radical labels. It is demonstrated that this technique can be used to noninvasively measure tissue oxygen concentration. In addition, rapid scan EPR measurements can be performed enabling gated millisecond measurements of oxygen concentrations to be performed over the cardiac cycle. Thus, low-frequency EPR spectroscopy offers great promise in the study of tissue oxygen concentrations and the role of oxygen in metabolic control.     

Electron transport to nitrogenase in Rhodospirillum rubrum: the role of NAD(P)H as electron donor and the effect of fluoroacetate on nitrogenase activity

The role of the reactions of the TCA cycle in the generation of reductant for nitrogenase in Rhodospirillum rubrum has been investigated. Addition of fluoroacetate inhibited nitrogenase activity almost completely when pyruvate or endogenous sources were used as electron donors, whereas the inhibition was incomplete when malate, succinate or fumarate were used. Addition of NAD(P)H to cells supported nitrogenase activity, both with and without prior addition of fluoroacetate. We suggest that the role of the TCA cycle in nitrogen fixation in R. rubrum is to generate reduced pyridine nucleotides which are oxidized by the components of the electron transport pathway to nitrogenase.     

Membrane penetration of nitric oxide and its donor S-nitroso-N-acetylpenicillamine: a spin-label electron paramagnetic resonance spectroscopic study

S-nitroso-N-acetylpenicillamine (SNAP) is a pharmacological agent with diverse biological effects that are mainly attributable to its favorable characteristics as a nitric oxide (NO)-evolving agent. It is found that SNAP incorporates readily into dimyristoyl phosphatidylcholine (DMPC) bilayer membranes; and an approximate penetration profile was obtained from the depth dependence of the perturbation that it exerts on spin-labeled lipid chains. The profile of SNAP locates it deep in the hydrophobic core of both fluid- and gel-phase membranes. The spin relaxation enhancement of spin-labeled phospholipids with nitroxide group located at different depths in DMPC membranes was determined for nitric oxide (NO) and molecular oxygen (O₂), at close to atomic spatial resolution. The relaxation enhancement, which is proportional to the corresponding vertical membrane profile of the concentration-diffusion product, was measured in the gel and fluid phases of the lipid bilayer. No significant membrane penetration was observed in the gel phase for the two water-dissolved gases. In the fluid phase, the transmembrane profiles of NO and O₂ are similar and could be well described by a sigmoidal function with a maximum in the center of the bilayer, but that of NO is less steep and is shifted toward the center of the membrane, relative to that of O₂. These differences can be attributed mainly to the difference in hydrophobicity between the two gases and the presence of the donor in the NO experiments. The biological implications of the above results are discussed.     

Direct superoxide anion scavenging by a disodium disuccinate astaxanthin derivative: Relative efficacy of individual stereoisomers versus the statistical mixture of stereoisomers by electron paramagnetic resonance imaging

Carotenoids are a related group of greater than 600 natural compounds, irrespective of geometric- and stereoisomers, with demonstrated antioxidant efficacy. The carotenoids are broadly divided into "carotenes," or non-oxygen substituted hydrocarbon carotenoids, and "xanthophylls," oxygen-substituted carotenoids. The natural compounds are excellent singlet oxygen quenchers as well as lipid peroxidation chain-breakers; this dual antioxidant capacity is generally attributed to the activity of the polyene chain, and increases with the number of conjugated double bonds along the polyene chain length. However, the poor aqueous solubility of most carotenes and the vast majority of xanthophylls limits their use as aqueous-phase singlet oxygen quenchers and direct radical scavengers. A variety of introduction vehicles (e.g., organic solvents, cyclodextrins) have been used to introduce the insoluble carotenoids into aqueous test systems. Hawaii Biotech, Inc. (HBI) successfully synthesized a novel carotenoid derivative, the disodium disuccinate derivative of astaxanthin (3,3(')-dihydroxy-beta,beta-carotene-4,4(')-dione) in all-trans (all-E) form. The novel derivative is a water-dispersible symmetric chiral molecule with two chiral centers, yielding four stereoisomeric forms: 3R,3(')R and 3S,3(')S (enantiomers), and the diastereomeric meso forms (3R,3(')S and 3(')R,3S). The individual stereoisomers were synthesized at high purity (>90% by HPLC) and compared directly for efficacy with the statistical mixture of stereoisomers obtained from the synthesis from the commercial source of astaxanthin (1:2:1 ratio of 3S,3(')S, meso, and 3R,3(')R, respectively). Direct scavenging of superoxide anion was evaluated in a standard in vitro isolated human neutrophil assay by electron paramagnetic resonance (EPR) imaging, employing the spin-trap DEPMPO. Each novel derivative was tested in pure aqueous formulation and in ethanolic formulation shown to completely disaggregate the compounds in solution. In each case, the ethanolic formulation was a more potent scavenging vehicle. No significant differences in scavenging efficiency were noted among the individual stereoisomers and the statistical mixture of stereoisomers, suggesting that the polyene chain alone was responsible for superoxide scavenging. Dose-ranging revealed that the statistical mixture of stereoisomers of the novel derivative, at millimolar (mM) concentrations, could nearly completely eliminate the superoxide anion signal generated in the activated human neutrophil assay. All ethanolic formulations of the novel derivatives exhibited increased scavenging efficiency over equimolar concentrations of non-esterified astaxanthin delivered in a dimethyl sulfoxide (DMSO) vehicle. These novel compounds will likely find utility in applications requiring aqueous delivery of a highly potent direct radical scavenger.     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.     

Biological effects of cigarette smoke, wood smoke, and the smoke from plastics: The use of electron spin resonance

This review compares and contrasts the chemistry of cigarette smoke, wood smoke, and the smoke from plastics and building materials that is inhaled by persons trapped in fires. Cigarette smoke produces cancer, emphysema, and other diseases after a delay of years. Acute exposure to smoke in a fire can produce a loss of lung function and death after a delay of days or weeks. Tobacco smoke and the smoke inhaled in a burning building have some similarities from a chemical viewpoint. For example, both contain high concentrations of CO and other combustion products. In addition, both contain high concentrations of free radicals, and our laboratory has studied these free radicals, largely by electron spin resonance (ESR) methods, for about 15 years. This article reviews what is known about the radicals present in these different types of smokes and soots and tars and summarizes the evidence that suggests these radicals could be involved in cigarette-induced pathology and smoke-inhalation deaths. The combustion of all organic materials produces radicals, but (with the exception of the smoke from perfluoropolymers) the radicals that are detected by ESR methods (and thus the radicals that would reach the lungs) are not those that arise in the combustion process. Rather they arise from chemical reactions that occur in the smoke itself. Thus, a knowledge of the chemistry of the smoke is necessary to understand the nature of the radicals formed. Even materials as similar as cigarettes and wood (cellulose) produce smoke that contains radicals with very different lifetimes and chemical characteristics, and mechanistic rationales for this are discussed. Cigarette tar contains a semiquinone radical that is infinitely stable and can be directly observed by ESR. Aqueous extracts of cigarette tar, which contain this radical, reduce oxygen to superoxide and thus produce both hydrogen peroxide and the hydroxyl radical. These solutions both oxidize alpha-1-proteinase inhibitor (a1PI) and nick DNA. Because of the potential role of radicals in smoke-inhalation injury, we suggest that antioxidant therapy (such as use of an inhaler for persons brought out of a burning building) might prove efficacious.