Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.     

On the interpretation of electron paramagnetic resonance spectra of binuclear iron-sulfur proteins

A method is described for the interpretation of electron paramagnetic resonance spectra of reduced binuclear iron-sulfur proteins. The g_y values for any protein can be analyzed so that both the symmetry and the extent of covalency at the paramagnetic site can be parameterized. These parameters can be related to the chemical composition of the paramagnetic center, the protein-dependent charge delocalization of the unpaired electron, and the geometric arrangement at the reduced iron atom. These analyses may ultimately be used to rationalize certain aspects of the redox potentials of the various iron-sulfur proteins.     

An electron paramagnetic resonance investigation of the oxygen dependence of the arterial-venous gradient of nitrosyl hemoglobin in blood circulation

Whether there is a nitrosyl hemoglobin (HbNO) gradient between the venous and the arterial parts of the circulatory system is a very controversial issue in nitric oxide research. We have carefully evaluated the measurement of HbNO concentration in blood using EPR generated in vivo by the NO donor DEANO under various oxygen tensions. We found that the absolute concentrations of HbNO in venous and arterial blood were the same within experimental error, independent of hemoglobin saturation; only the ratios of 5-coordinate and 6-coordinate HbNO differed. The HbNO concentration increased when the oxygen concentration breathed by the rats decreased in a manner that was linear in hemoglobin saturation. These results do not support the existence of an arterial-venous gradient of HbNO under our experimental conditions.     

35-GHz (Q-band) saturation transfer electron paramagnetic resonance studies of rotational diffusion

The extension of saturation transfer electron paramagnetic resonance spectroscopy (ST-EPR) to an observational frequency of 35 GHz (Q band) is described. At this frequency the spectral resolution is greatly enhanced over that afforded at the 9.5-GHz (X-band) frequency used in most of the ST-EPR studies published to date. Thus, Q-band operation may provide an approach for the detailed analysis of the slow anisotropic motions believed to occur in many biomolecular systems. The spectral characteristics and the effects of various instrumental settings are described in detail for a model system of spin-labeled hemoglobin in water-glycerol solutions. Several spectral parameters are defined, and their potential use in monitoring various types of anisotropic motion is considered.     

X- and Q-band electron paramagnetic resonance of CO2- in hydroxyapatite single crystals

Both X- and Q-band electron paramagnetic resonance (EPR) research has been conducted using slightly carbonated hydroxyapatite (HAp) single crystals after exposure to ionizing radiation. Below a temperature of 90 K, O(-) and CO(2-) radicals were detected, whereas at room temperature only CO(2-) spectra could be observed. The O(-) ion has previously been investigated in high-purity HAp single crystals, whereas EPR spectra of CO(2-) in HAp single crystals have not been reported. Both paramagnetic defects exhibit EPR angular variations in planes containing the c axis of the crystal from which spin Hamiltonian parameters were derived. Arguments are given for the presence of two CO(2-) defects in the irradiated HAp single crystals.     

Integer-spin electron paramagnetic resonance of iron proteins.

A quantitative interpretation is presented for EPR spectra from integer-spin metal centers having large zero-field splittings. Integer-spin, or non-Kramers, centers are common in metalloproteins and many give EPR signals, but a quantitative understanding has been lacking until now. Heterogeneity of the metal's local environment will result in a significant spread in zero-field splittings and in broadened EPR signals. Using the spin Hamiltonian Hs = S.D.S + beta S.g.B and some simple assumptions about the nature of the zero-field parameter distributions, a lineshape model was devised which allows accurate simulation of single crystal and frozen solution spectra. The model was tested on single crystals of magnetically dilute ferrous fluosilicate. Data and analyses from proteins and active-site models are presented with the microwave field B1 either parallel or perpendicular to B. Quantitative agreement of observed and predicted signal intensities is found for the two B1 orientations. Methods of spin quantitation are given and are shown to predict an unknown concentration relative to a standard with known concentration. The fact that the standard may be either a non-Kramers or a Kramers center is further proof of the model's validity. The magnitude of the splitting in zero magnetic field is of critical importance; it affects not only the chance of signal observation, but also the quantitation accuracy. Experiments taken at microwave frequencies of 9 and 35 GHz demonstrate the need for high-frequency data as only a fraction of the molecules give signals at 9 GHz.     

Electron paramagnetic resonance study of nitrosyl haemoglobin M Iwate

The nitrosyl derivative of haemoglobin M Iwate, a haemoglobin variant which has functional beta chains and non-functional alpha chains, is known to exist only in a low affinity form. It shows a nine-line superhyperfine (SHF) pattern in the low temperature electron paramegnetic resonance (e.p.r.) spectrum, even in the presence of inositol hexaphosphate. This provides further support for the suggestion (Chevion, M., Stern, A., Peisach, J., Blumberg, W. E. and Simon, S. Biochemistry 1978 bd17, 1745) that the beta chains of tetrameric, fully ligated, nitrosyl derivatives of all haemoglobins invariably exhibit an e.p.r. spectrum with a nine-line SHF pattern regardless of the affinity state of the molecule. On the other hand, nitrosyl ferrous alpha chains within the haemoglobin tetramer can exhibit an e.p.r. spectrum with either a nine-line or a three-line SHF pattern which is dependent upon the affinity state of the molecule.     

Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins

An extension of a method relating chemical structure to the EPR parameters A_∥ and g_∥ is presented. For complexes having the same atoms of ligation, a decrease in charge of the metal-ligand complex decreases g_∥ and increases A_∥. From this analysis, one concludes that in artificial copper proteins as well as in the naturally occurring nonblue copper proteins copper is ligated to oxygen and nitrogen but not to sulfur. A method is presented for the interpretation of EPR changes that occur with ligand exchange reactions at the Type 2 (nonblue) copper sites such as occur in laccase.     

Q-band electron paramagnetic resonance dosimetry in tooth enamel: biopsy procedure and determination of dose detection limit

High-frequency Q-band (37 GHz) electron paramagnetic resonance (EPR) dosimetry allows to perform fast (i.e., measurement time <15 min) dose measurements using samples obtained from tooth enamel mini-biopsy procedures. We developed and tested a new procedure for taking tooth enamel biopsy for such dose measurements. Recent experience with EPR dose measurements in Q-band using mini-probes of tooth enamel has demonstrated that a small amount of tooth enamel (2–10 mg) can be quickly obtained from victims of a radiation accident. Accurate dose assessments can further be carried out in a very short time to provide important information for medical treatment. Here, the Q-band EPR dose detection limit for 5 and 10 mg samples is estimated to be 367 and 248 mGy, respectively. These values are comparable to the critical parameters determined for conventional X-band EPR in tooth enamel.     

Electron spin relaxation of the electron paramagnetic resonance spectra of cytochrome c

The progressive power saturation of the electron paramagnetic resonance (EPR) spectrum of ferricytochrome c has been investigated in order to determine the spin-lattice relaxation time of the center. We have generalized the usual saturation treatments to include the effects of extended sample size and anisotropic g values as well as derivative spectra. We find that the results are consistent with a T7 power law in the temperature range 6--25 K. At temperatures above 25 K the relaxation time is too short for successful power saturation. Observation of the linewidth shows that the relaxation behavior continues as a first-order Raman process to 50 K.     

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.     

Calculation of electron paramagnetic resonance spectra from Brownian dynamics trajectories: application to nitroxide side chains in proteins.

We present a method to simulate electron paramagnetic resonance spectra of spin-labeled proteins that explicitly includes the protein structure in the vicinity of the attached spin label. The method is applied to a spin-labeled polyleucine alpha-helix trimer. From short (6 ns) stochastic dynamics simulations of this trimer, an effective potential energy function is calculated. Interaction with secondary and tertiary structures determine the reorientational motion of the spin label side chains. After reduction to a single particle problem, long stochastic dynamic trajectories (700 ns) of the spin label side-chain reorientation are calculated from which the Lamor frequency trajectory and subsequently the electron paramagnetic resonance spectrum is determined. The simulated spectra agree well with experimental electron paramagnetic resonance spectra of bacteriorhodopsin mutants with spin labels in similar secondary and tertiary environments as in the polyleucine.     

Variant MoFe proteins of Azotobacter vinelandii: effects of carbon monoxide on electron paramagnetic resonance spectra generated during enzyme turnover

The resting state of wild-type nitrogenase MoFe protein exhibits an S=3/2 electron paramagnetic resonance (EPR) signal originating from the FeMo cofactor, the enzymes active site. When nitrogenase turns over under CO, this signal disappears and one (sometimes two) of three new EPR signals, which also arise from the FeMo cofactor, appears, depending on the CO concentration. The appearance and properties of these CO-inducible EPR signals, which were also generated with variant MoFe proteins (R96Q, R96K, Q191K, R359K, R96K/R359K, R277C, R277H, and nifV) that are impacted around the FeMo cofactor, have been investigated. No new CO-induced EPR signals arise from any variant, suggesting that no new CO-binding sites are produced by the substitutions. All variant proteins, except R277H, produce the lo-CO signal; all, except Q191K, produce the hi(5)-CO signal; but only two (R96Q and nifV) exhibit the hi-CO signal. FeMo cofactors environment clearly dictates which CO-induced EPR signals are generated; however, none of these EPR signals correlate with CO inhibition of H₂ evolution observed with some of these variants. CO inhibition of H₂ evolution is, therefore, due to CO binding to a different site(s) from those responsible for the CO-induced EPR signals. Some resting-state variants have overlapping S=3/2 EPR signals, whose intensities simultaneously decrease under turnover conditions, indicating that all FeMo cofactor conformations are catalytically active. Moreover, these variants produce a similar number of hi(5)-CO signals after turnover under CO to the number of resting-state S=3/2 signals. The FeMo cofactor associated with the hi(5)-CO signal likely contains two bridging CO molecules.     

Electron-nuclear coupling in nitrosyl heme proteins and in nitrosyl ferrous and oxy cobaltous tetraphenylporphyrin complexes

Electron spin echo envelope modulation (ESEEM) spectroscopy has been used to study electron-nuclear interactions in the following isoelectronic S = 1/2 complexes: NO-FeII(TPP) (TPP = tetraphenylporphyrin) with and without axial nitrogenous base, nitrosylhemoglobin in R and T states, and O2-CoII(TPP) with and without axial base. Only the porphyrin pyrrole nitrogens contribute to the ESEEM of the 6-coordinate nitrosyl FeII(TPP) complexes, nitrosylhemoglobin (R-state), and the nitrosyl complexes of alpha and beta chains. Pyrrole nitrogens in the 5-coordinate complex NO-FeII(TPP) are coupled too weakly to unpaired spin and therefore do not contribute to the ESEEM. A partially saturated T-state nitrosylhemoglobin does not exhibit echo envelope modulations characteristic of 6-coordinate nitrosyl species, which confirms that the proximal imidazole bond to heme iron is disrupted. Study of 6-coordinate O2-CoII(TPP)(L) complexes (L = nitrogenous base) using 14N- and 15N-labeled ligands and porphyrins enabled a detailed analysis of coupling parameters for both pyrrole and axial nitrogens. The pyrrole 14N coupling frequencies are similar to those in NO-FeII(TPP)(L). The Fermi contact couplings for axially bound nitrogen, calculated from simulation of ESEEM spectra for a series of O2-CoII(TPP)(L) complexes (L = pyridine, 4-picoline, 4-cyanopyridine, 4-carboxypyridine, and 1-, 2-, and 4-methylimidazole) illustrate a trend toward stronger hyperfine interactions with weaker bases.