ENDOR from nitrogens and protons in low spin ferric heme and hemoprotein.

Electron nuclear double resonance (ENDOR) signals have been obtained from iron-linked nitrogens in frozen solutions of cytochrome c, metmyoglobin cyanide, and a low spin protohemin mercaptide complex. Hyperfine couplings from heme protons have also been obtained from metmyoglobin cyanide and from a low spin protohemin cyanide complex. Several of these proton resonances are assigned to specific heme protons.

Electron nuclear double resonance of cytochrome oxidase: nitrogen and proton ENDOR from the 'copper' EPR signal.

Proton ENDOR resonances have been found from at least two different protons with fairly large and isotropic couplings of about 12 and 19 MHz. It is possible that such protons are attached to carbons that are one bond removed from the point of ligation to copper. A number of weakly coupled protons with anisotropic couplings have also been seen. None of the protons, either weakly or strongly coupled, appears to exchange with 2H2O. We have obtained nitrogen ENDOR from at least one nitrogen with a hyperfine coupling large enough for the nitrogen to be a ligand of copper. We have not yet demonstrated experimentally ENDOR characteristic of the copper nucleus itself.

Two-electron reduction of cytochrome c oxidase triggers a conformational transition.

The slow increase of a cyanide-induced optical change at 437 nm following rapid cyanide inhibition of cytochrome oxidase has been followed as a function of the number of electrons donated from ferrocytochrome c to cytochrome a and CuA. The initial rate of optical change is a parabolic function of this number. The results have been analyzed in terms of a model where addition of electrons causes a conformational transition allowing rapid cyanide binding. The binding is followed by a slow intramolecular responsible for the optical change. The analysis demonstrates that only molecules with both cytochrome a and CuA reduced can undergo the conformational change, which is suggested to be involved in the proton-pump mechanism of the oxidase.

Double-stacked dielectric resonator for sensitive EPR measurements.

A new approximate method for predicting the resonant frequencies and for solving the field distribution problem of a cylindrical dielectric resonator (DR) is developed. The model proposed in this paper bridges the gap between rigorous and accurate finite-element or Green function-based numerical methods on the one hand and on the other hand, simple approximate solutions in which the field distribution can be described analytically, but the resulting frequency is accurate within a few percent only. In the method described here, the approximate solution for the microwave field distribution is modified by substituting different values of the radial separation constants inside and outside of the diskshaped DR. The model is generalized for the double-stacked DR structure and enables one to introduce corrections that take into account the presence of the shielding walls and of the cylindrical sample hole. Good agreement is found between experimental and calculated results for both the single and double-stacked structures that are designed around commercially available X-band DRs (9-10 GHz). For the resonant frequency of the lowest transverse-electric TEzero1 delta mode that is commonly used for EPR measurements, the accuracy of the method is better than 1%. Experimentally measured resonator filling factors are also in good agreement with those theoretically estimated. Both the theory and the experimental results suggest that the double-stacked DR structure with finite spacing between the ceramic cylinders is the most suitable for EPR measurements of long lossy samples.

Dielectric resonator-based flow and stopped-flow EPR with rapid field scanning: A methodology for increasing kinetic information.

We report methodology which combines recently developed dielectric resonator-based, rapid-mix, stopped-flow EPR (appropriate for small, aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic field where the scanning is preprogrammed to occur at selected times after the start of flow. This methodology gave spectroscopic information complementary to that obtained by stopped-flow EPR at single fields, and with low reactant usage, it yielded more graphic insight into the time evolution of radical and spin-labeled species. We first used the ascorbyl radical as a test system where rapid scans triggered after flow was stopped provided "snapshots" of simultaneously evolving and interacting radical species. We monitored ascorbyl radical populations either as brought on by biologically damaging peroxynitrite oxidant or as chemically and kinetically interacting with a spectroscopically overlapping nitroxide radical. In a different biophysical application, where a spin-label lineshape reflected rapidly changing molecular dynamics of folding spin-labeled protein, rapid scan spectra were taken during flow with different flow rates and correspondingly different times after the mixing-induced inception of protein folding. This flow/rapid scan method is a means for monitoring early immobilization of the spin probe in the course of the folding process.

Dielectric resonator-based side-access probe for muscle fiber EPR study.

We present a novel dielectric resonator (DR)-based resonant structure that accommodates aqueous sample capillaries in orientations that are either parallel (i.e., side-access) or perpendicular to the direction of an external (Zeeman) magnetic field, B(0). The resonant structure consists of two commercially available X-band DRs that are separated by a Rexolite spacer and resonate in the fundamental TE(01delta) mode. The separator between the DRs is used to tune the resonator to the desired frequency and, by appropriately drilled sample holes, to provide access for longitudinal samples, notably capillaries containing oriented, spin-labeled muscle fibers. In contrast to the topologically similar cylindrical TE(011) cavity, the DR-based structure has distinct microwave properties that favor its use for parallel orientation of lossy aqueous samples. For perpendicular orientation of a dilute (6.25 microM) aqueous solution of IASL spin label, the S/N ratio was at least one order of magnitude better for the side-access DR-based structure than for a standard TE(102) cavity. EPR spectra acquired for maleimide spin-labeled myosin filaments also revealed ca. 10 times better S/N ratio than those obtained with a standard TE(102) cavity. For the side-access DR with sample capillaries oriented either parallel or perpendicular to the external magnetic field, the Q- and filling factors are in good agreement with the theoretical estimates derived from the distribution of magnetic (H(1)) and electric (E(1)) components.

Hyperfine structure from the four pyrrole nitrogens of heme.

Hemin and hematin have been incorporated into single crystals of the condensed-ring planar hydrocarbon perylene, the approximate molecular ratio of heme to aromatic being 10(-3). X-ray diffraction and electron paramagnetic resonance (EPR) measurements indicate that these mixed crystals maintain their integrity when subjected to cryogenic temperatures and then returned to room temperature. EPR X-band orientation studies of single crystals at liquid helium temperature have revealed the following two important features: (1) presence of ligand hyperfine structure, which can be used to quantitate the distribution of unpaired metal electron on the ligands and (2) removal of the immediate fourfold symmetry of the ligand field about the metal, as evidenced by the rhombic character of the high-spin ferric system. The ligand hyperfine splittings have been interpreted as arising chiefly from an isotropic Fermi contact interaction. The observed splittings then indicate that 2.7 per cent of an unpaired spin is in each nitrogen 2s orbital. The g-values have been interpreted in terms of the spin-Hamiltonian: [Formula: see text] which gives g(x,y) = 6.00 +/- 24E/D and g(z) = 2.00 - 34(E/D)(2). For various hemin and hematin sites these formulae for the g-values are nicely obeyed when E/D is in the range 0.015 to 0.03.

Variable velocity liquid flow EPR applied to submillisecond protein folding.

We have developed a variable velocity, rapid-mix, continuous-flow method for observing and delineating kinetics by dielectric resonator-based electron paramagnetic resonance (EPR). The technology opens a new facet for kinetic study of radicals in liquid at submillisecond time resolution. The EPR system (after Sienkiewicz, A., K. Qu, and C. P. Scholes. 1994. Rev. Sci. Instrum. 65:68-74) accommodated a miniature quartz capillary mixer with an approximately 0.5 microliter delivery volume to the midpoint of the EPR-active zone. The flow velocity was varied in a preprogrammed manner, giving a minimum delivery time of approximately 150 microseconds. The mixing was efficient, and we constructed kinetics in the 0.15-2. 1-ms time range by plotting the continuous wave EPR signal taken during flow versus the reciprocal of flow velocity. We followed the refolding kinetics of iso-1-cytochrome c spin-labeled at Cysteine 102. At 20 degrees C, upon dilution of guanidinium hydrochloride denaturant, a fast phase of refolding was resolved with an exponential time constant of 0.12 ms, which was consistent with the "burst" phase observed by optically detected flow techniques. At 7 degrees C the kinetic refolding time of this phase increased to 0.5 ms.

Endonuclease III interactions with DNA substrates. 1. Binding and footprinting studies with oligonucleotides containing a reduced apyrimidinic site.

The binding of endonuclease III from Escherichia coli to damaged DNA has been studied using gel shift and footprinting assays. Oligonucleotides containing a reduced apyrimidinic (AP) site were used since reduction of the AP site blocks the beta-elimination reaction catalyzed by the enzyme and yields a noncleavable substrate. The Kobs for a 13-mer carrying a centrally located reduced AP site is (2 x 10(6)-(2 x 10(7) M-1, while the Kobs for a 13-mer with no damage is (4.5 x 10(3)-(3.2 x 10(4) M-1 (approximately a 500-fold difference). Larger oligonucleotides would not enter a gel when endonuclease III was bound so that binding constants to oligonucleotides longer than 13 base pairs could not be determined directly. Competition assays suggest that the Kobs measured for both damaged and undamaged 13-mers is a minimum value and that the Kobs for larger oligonucleotides could be an order of magnitude greater. Fluorescence quenching on related 19-mers yielded a specific binding constant for the 19-mer carrying a centrally located reduced AP site for 4 x 10(7) M-1 and a nonspecific binding constant to an undamaged 19-mer of approximately 10(5) M-1 [Xing, D., Dorr, R., Cunningham, R. P., & Scholes, C. P. (1995) Biochemistry 34, 2537-2544]. Several footprinting reagents were used to determine the size and location of the endonuclease III binding site on damaged oligonucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

The identification of histidine ligands to cytochrome a in cytochrome c oxidase.

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.

On the nature of cysteine coordination to CuA in cytochrome c oxidase.

The resolution of new features in the 1H electron nuclear double resonance (ENDOR) spectrum of the oxidized CuA site in beef heart cytochrome c oxidase is presented. In a previous study, we assigned resonances in the CuA ENDOR spectrum to hyperfine interactions of methylene protons on one or two cysteine ligands to CuA (Stevens, T.H., Martin, C.T., Wang, H., Brudvig, G.W., Scholes, C.P., and Chan, S.I. (1982) J. Biol. Chem. 257, 12106-12113). In this work, a new 1H ENDOR resonance in the beef heart CuA ENDOR spectrum is reported and can be assigned to either anisotropy in a previously resolved cysteine methylene proton hyperfine interaction (Aiso = 12 MHz, Aaniso = 2.5 MHz) or to a third isotropic hyperfine coupling (A = 13.6 MHz) to a cysteine methylene proton of a second cysteine ligand to copper. In either case, the 1H ENDOR results require the delocalization of approximately 50% of the unpaired spin from copper onto either one or two cysteine ligands to CuA. To characterize further the CuA site, we have prepared yeast cytochrome c oxidase incorporating isotopically substituted [beta-13C]cysteine. The CuA ENDOR spectrum of this species shows only one clearly resolved 13C hyperfine interaction (A = 3.6 MHz). This result confirms the assignment of at least one strongly interacting cysteine ligand to CuA and suggests that if the assignment of two cysteine ligands to CuA is correct, the two cysteines interact with copper in a highly symmetric manner. A recent extended x-ray absorption fine structure study of native and modified forms of cytochrome c oxidase indicates the coordination of two sulfur ligands to CuA (Li, P.M., Gelles, J., Chan, S.I., Sullivan R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095). In light of the new possibility of two symmetrically coordinated cysteine ligands to CuA, we propose a molecular orbital description of the oxidized CuA site which is characterized by a high degree of delocalization of unpaired spin away from copper and onto a pair of symmetrically coordinated cysteine sulfur ligands. We also present a protein model for the CuA site in which two cysteine ligands derived from subunit II lie on the face of an alpha-helix. This structure would allow the unprecedented stable coordination of two cysteine thiolate sulfurs to copper and may provide a mechanism for the redox-linked proton pumping by cytochrome c oxidase.

Electron nuclear double resonance study of the Mn2+ environs in the oxalate-ATP complex of pyruvate kinase.

Electron nuclear double resonance (ENDOR) and the related pulse technique of pulse field sweep EPR (PFSEPR) were used to probe the site I environment of Mn2+ in the oxalate-ATP complex of pyruvate kinase. Assignment of features and an estimate of hyperfine couplings have shown proximity of protons to the metal ions through their dipolar interaction and proximity of 31P and 17O because of a contact interaction from direct Mn(2+)-ligand covalent spin transfer. Since Mn2+ is a spin5/2 ion whose Ms = +/- 1/2, +/- 3/2, and +/- 5/2 electron spin states can all contribute to EPR and ENDOR, we have developed experimental and theoretical strategies for elucidating hyperfine couplings to the Mn2+ electron spin states. Solvent-exchangeable proton ENDOR features were evident with couplings very similar to the hyperfine couplings of H2O in [Mn(H2O)6]2+. ENDOR of exchangeable, more distant protons originated from a dipolar coupling such as could be expected from protons residing 5.5 A from Mn2+ and hydrogen-bonded to a nonliganding oxygen or nitrogen. Nonexchangeable proton ENDOR features indicated dipolar coupling to proton(s) from the protein residing at approximately 4.5 A from the Mn2+. The approximately 4-MHz 31P phosphate hyperfine couplings in Mn(II)-nucleotide models and in pyruvate kinase were similar, but a detailed ENDOR and PFSEPR comparison revealed that the hyperfine coupling to the ATP gamma-phosphate in pyruvate kinase was approximately 10% less than coupling to phosphates of Mn(II)-nucleotides. [In pyruvate kinase only the gamma-phosphate has been shown to bind to Mn2+ at site I (Lodato & Reed, 1987).](ABSTRACT TRUNCATED AT 250 WORDS)

The method of time-resolved spin-probe oximetry: its application to oxygen consumption by cytochrome c oxidase.

This work broadens the scope and improves the time resolution of spin-probe oximetry, a technique in which small nitroxide spin probes detect oxygen consumption via change in their relaxation properties [Froncisz, W., Lai, C.-S., & Hyde, J. S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 411-415]. For rapid oxygen kinetic studies we combined the methodology of spin-probe oximetry with a recently developed loop-gap resonator, stopped-flow EPR system [Hubbell, W. L., Froncisz, W., & Hyde, J. S. (1987) Rev. Sci. Instrum. 58, 1879-1886]. The technique used microliter volumes of reactant solutions. Enzymatic consumption of oxygen by cytochrome c oxidase in the presence of ferrocytochrome c substrate was followed continuously in time under limited-turnover conditions, where the concentration of oxygen consumed often was comparable to or less than the amount of enzyme present. In detecting less than micromolar oxygen concentration changes, we have achieved a time resolution of the order 30 ms when flow is stopped. Oxygen consumption was followed under two different limited-turnover conditions: In the first, the amount of oxygen consumed was limited by available ferrocytochrome c, and the time course of oxygen consumption and its pH dependence were compared with the optically detected ferrocytochrome c consumption. In the second, the oxygen consumed was ultimately limited by the availability of oxygen itself while ferrocytochrome c was regenerated and remained in excess.(ABSTRACT TRUNCATED AT 250 WORDS)

Endonuclease III interactions with DNA substrates. 2. The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching.

We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs. Endonuclease III has two tryptophans, Trp132 in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron-sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O'Handley, S. F., & Cunningham, R. P. (1992) Science 258, 434-440]. The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment. Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are differently accessible to anionic quencher. Significant (approximately 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weight duplex undamaged poly(dAdT). The apparent second-order nonspecific binding constant to poly(dAdT) was 4 x 10(7) M-1, and there were approximately 12 base pairs per endonuclease III binding site for binding to poly(dAdT). This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA. A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was approximately 400-fold larger than to the undamaged 19-mer.(ABSTRACT TRUNCATED AT 250 WORDS)

EPR-detected folding kinetics of externally located cysteine-directed spin-labeled mutants of iso-1-cytochrome c.

We report the application of our newly developed dielectric resonator-based flow and stopped-flow kinetic EPR systematically to probe protein folding in yeast iso-1-cytochrome c at cysteine-directed spin-labeled locations. The locations studied have not been previously directly probed by other techniques, and we observe them on a time scale stretching from 50 micros to seconds. On the basis of crystal structure and homology information, the following mutation-tolerant, externally located cysteine labeling sites were chosen (in helices, T8C, E66C, and N92C; in loops, E21C, V28C, H39C, D50C, and K79C), and labeling at these sites was not destabilizing. Dilution of denaturant was used to induce folding and thereby to cause a change in the spin label EPR signal as folding altered the motion of the spin label. Under folding conditions, including the presence of imidazole to eliminate kinetic trapping due to heme misligation, a phase of folding on the 20-30 ms time scale was found. This phase occurred not only at the T8C and N92C labeling sites in the N- and C-terminal helices, where such a phase has been associated with folding in these helices, but overall at labeling sites throughout the protein. In the absence of imidazole the 20-30 ms phase disappeared, and another phase having the time scale of 1 s appeared throughout the protein. There was evidence under all conditions for a burst phase on a scale of less than several milliseconds which occurred at labeling positions V28C, H39C, D50C, E66C, and K79C in the middle of the protein sequence. At spin-labeled D50C rapid-mix flow EPR indicated a very short approximately 50 micros phase possibly associated with the prefolding or compaction of the loop to which D50 belongs. Spin labels have been criticized as perturbing the phenomena which they measure, but our spin labeling strategy has reported common kinetic themes and not perturbed, disconnected kinetic events.

Electronic structural information from Q-band ENDOR on the type 1 and type 2 copper liganding environment in wild-type and mutant forms of copper-containing nitrite reductase.

Q-band ENDOR elucidated proton and nitrogen hyperfine features to provide spin density information at ligands of blue-green Type 1 and catalytic Type 2 copper centers in nitrite reductase. The blue-green Type 1 center of nitrite reductase has a redox, electron-transfer role, and compared to the blue center of plastocyanin, it has the following structural differences: a shortened Cu-Smet bond length, a longer Cu-Scys bond length, and altered ligand-copper-ligand bond angles (Adman, E. T., Godden, J. W., and Turley, S. (1995) J. Biol. Chem. 270, 27458-27474). The hyperfine couplings of the two Type 1 histidine (N delta) ligands showed a larger percentage difference from each other in electron spin density than previously reported for other blue Type 1 proteins, while the cysteine beta-proton hyperfine couplings, a measure of unpaired p pi spin density on the liganding cysteine sulfur, showed a smaller electron spin density. A mutation of the Type 1 center, M182T, having the copper-liganding Met182 transformed to Thr182, caused the center to revert to an optically "blue" center, raised its redox potential by approximately 100 mV, and led to the loss of activity (prior paper). Surprisingly, in M182T there was no change from native Type 1 copper either in the histidine or cysteine hyperfine couplings or in g values and Cu nuclear hyperfine couplings. The conclusion is that the optical and redox alterations due to changed Type 1 methionine ligation need not be concurrent with electron spin delocalization changes in the HOMO as reported from its essential cysteine and histidines. A detailed picture of the nitrogen couplings from the three histidine (N epsilon) ligands of the Type 2 center indicated a substantial ( approximately 200%) electronic hyperfine inequivalence of one of the histidine nitrogens from the other two within the Type 2 HOMO and thus provided evidence for electronic distortion of the Type 2 site. In the presence of the nitrite substrate, hyperfine couplings of all histidines diminished. We suggest that this nitrite-induced decreased covalency would correlate with an increased Type 2 redox potential to assist electron transfer to the Type 2 center. Dipole-coupled, angle-selected exchangeable proton features, observed over a range of g values, predicted a ligand-water proton distance of 2.80 A from copper, and these water protons were eliminated by nitrite. His287 is not a Type 2 ligand but is positioned to perturb an axial water or a nitrite of Type 2 copper. In the presence of nitrite the mutant H287E showed no evidence for the loss of water protons and no diminished ligand histidine covalency. H287E has vastly diminished activity (prior paper), and the ENDOR information is that NO2- does not bind to Type 2 copper of H287E. In summary, the electronic information from this study of native and suitably chosen mutants provided a test of the highest occupied molecular orbital (HOMO) wave function at Type 1 and Type 2 coppers and an intimate electronic insight into functional enzymatic properties.