Continuous-wave Electron Nuclear Double Resonance Research Articles

Basic Molecular Unit Involved in Charge Migration in Oxidized Light-Harvesting Complex 1 of Rhodobacter sphaeroides

The light-harvesting complex 1(LH1) of bacterial photosynthesis harvests sunlight and transfers the energy to its inner reaction center (RC) core to drive the photosynthetic process. After chemical oxidation of the LH1 complexes of Rhodobacter sphaeroides, charge migration within the bacteriochlorophyll a (BChla) molecular array is observed at very low temperature, suggesting this protein complex as a paradigm for charge migration in the solid state and nanotechnology development. A fundamental question is whether the two BChla molecules of the α/β BChla2 structural building block function as an interacting unit for charge migration or as individual molecules. The basic charge-migration unit of LH1 of Rb. sphaeroides has been investigated by continuous-wave electron nuclear double resonance (ENDOR) at 10 K. From the hyperfine coupling constants of the unpaired electron with its neighboring atoms, the basic charge-migration unit explored by ENDOR is consistent only with a monomer of BChla.

ENDOR and ESEEM investigation of the Ni-containing superoxide dismutase

Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O2(-) to H(2)O(2) and O(2). The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band (14)N,(1/2)H continuous wave (CW) and pulsed electron-nuclear double resonance (ENDOR) and X-band (14)N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band (14)N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial N(delta) of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band (14)N ESEEM characterized the hyperfine coupling of N(epsilon) of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between N(epsilon)-H and O(Glu-17) present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band (1)H CW ENDOR and pulsed (2)H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine coupling to either the proton(s) of a water in the pocket at the side opposite the axial N(delta) or the proton of a water hydrogen-bonded to the equatorial thiolate ligand.

Structural basis for VO2+ inhibition of nitrogenase activity (A): 31P and 23Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy

We previously reported the vanadyl hyperfine couplings of VO(2+)-ATP and VO(2+)-ADP complexes in the presence of the nitrogenase Fe protein from Klebsiella pneumoniae (Petersen et al. in Biochemistry 41:13253-13263, 2002). It was demonstrated that different VO(2+)-nucleotide coordination environments coexist and are distinguishable by electron paramagnetic resonance (EPR) spectroscopy. Here orientation-selective continuous-wave electron-nuclear double resonance (ENDOR) spectra have been investigated especially in the low-radio-frequency range in order to identify superhyperfine interactions with nuclei other than protons. Some of these resonances have been attributed to the presence of a strong interaction with a 31P nucleus although no resolvable superhyperfine structure due to 31P or other nuclei was detected in the EPR spectra. The superhyperfine coupling component is determined to be about 25 MHz. Such a 31P coupling is consistent with an interaction of the metal with phosphorus from a directly, equatorially coordinated nucleotide phosphate group(s). Additionally, novel more prominent 31P ENDOR signals are detected in the low-frequency region. Some of these correspond to a relatively weak 31P coupling. This coupling is present with ATP for all pH forms but is absent with ADP. The ENDOR resonances of these weakly coupled 31P are likely to originate from an interaction of the metal with a nucleotide phosphate group of the nucleoside triphosphate and are attributed to a phosphorus with axial characteristics. Another set of resonances, split about the nuclear Zeeman frequency of 23Na, was detected, suggesting that a monovalent Na+ ion is closely associated with the divalent metal-nucleotide binding site. Na+ replacement by K+ unambiguously confirmed that ENDORs at radio frequencies between 3.0 and 4.5 MHz arise from an interaction with Na+ ions. In contrast to the low-frequency 31P signal, these resonances are present in spectra with both ADP and ATP, and for both low- and neutral-pH forms, although slight differences are detected, showing that these are sensitive to the nucleotide and pH.

A Multi-Frequency Pulse EPR and ENDOR Approach to Study Strongly Coupled Nuclei in Frozen Solutions of High-Spin Ferric Heme Proteins

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

An improved TM110 resonator for continuous-wave ENDOR studies at X-band

An improved TM110 resonator for continuous-wave and time-resolved electron-nuclear double resonance (ENDOR) studies at X-band frequencies is described that has been designed with small samples and/or light excitation in mind. The filling factor is increased by reducing the resonator length to only 16 mm. The radio-frequency field is generated by either a conventional solenoid for stable samples or by a pair of coils for samples which require photoexcitation. This arrangement leaves the central volume of the resonator free for optimal sample illumination, which is achieved through a slit in the wall of the resonator. Microwave coupling is achieved by the incorporation of an iris in the top of the resonator, and magnetic field modulation is applied by external coils. The resonator’s high sensitivity is illustrated by a study of the temperature dependence of the continuous-wave ENDOR signal of the stable neutral flavin radical in DNA photolyase and an investigation of the photoexcited triplet state of free-base tetraphenylbacteriochlorin by time-resolved ENDOR.

A continuous-wave electron-nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine beta-mono-oxygenase of the adrenal medulla.

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu(2+) centre with the following EPR parameters: g(z) 2.24, g(x,y) 2.06, A(Cu)(z) 19.0 mT and A(Cu)(x,y) 1.0 mT. Simulation of the Cu(2+) spectrum yielded a best match by using four equivalent nitrogens (A(N)=1.5 mT, 42 MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu(2+), in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron-nuclear double resonance (CW ENDOR; 9.4 GHz, 5-20 K) experiments at g( perpendicular) of the Cu(II) spectrum show a weak coupling to protons with an A(H) of 2.9 MHz that corresponds to a distance of 3.8 A (1 A identical with 0.1 nm), assuming that it is a purely dipolar coupling. Incubation in (2)H(2)O leads to a significant decrease in these (1)H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu(2+) by diethyldithiocarbamic acid. The (1)H and (14)N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu(2+) enzyme, dopamine beta-mono-oxygenase (DbetaM). For DbetaM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and (2)H(2)O-exchangeable protons (2.9 MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40 MHz) from the co-ordinating N of the histidines in DbetaM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

A continuous-wave electron–nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine β-mono-oxygenase of the adrenal medulla

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu2+ centre with the following EPR parameters: gz 2.24, gx,y 2.06, ACuz 19.0mT and ACux,y 1.0mT. Simulation of the Cu2+ spectrum yielded a best match by using four equivalent nitrogens (AN = 1.5mT, 42MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu2+, in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron—nuclear double resonance (CW ENDOR; 9.4GHz, 5–20K) experiments at g⊥ of the Cu(II) spectrum show a weak coupling to protons with an AH of 2.9MHz that corresponds to a distance of 3.8Å (1Å≡0.1nm), assuming that it is a purely dipolar coupling. Incubation in 2H2O leads to a significant decrease in these 1H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu2+ by diethyldithiocarbamic acid. The 1H and 14N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu2+ enzyme, dopamine β-mono-oxygenase (DβM). For DβM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and 2H2O-exchangeable protons (2.9MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40MHz) from the co-ordinating N of the histidines in DβM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

A Pulse EPR and ENDOR Investigation of the Electronic Structure of a σ-Carbon-Bonded Cobalt(IV) Corrole

In this contribution we present a continuous wave (CW), pulse electron paramagnetic resonance (EPR), and pulse electron nuclear double resonance (ENDOR) study of (OEC)Co(C6H5), where OEC is the trianion of 2,3,7,8,12,13,17,18-octaethylcorrole. To facilitate spectral assignments isotopic substitutions were employed (2H and 13C). From the analysis of the frozen solution CW EPR, ESEEM, and ENDOR spectra measured at X- and Q-band, we determined the electronic coupling parameters of the unpaired electron with the cobalt nucleus, corrole nitrogen nuclei, phenyl 13C, 1H and 2H nuclei, meso 1H and 2H nuclei, and ethyl 1H nuclei. Determination of the g matrix alignment in the molecular frame was achieved by successfully simulating the orientationally selective powder ENDOR spectra of the meso nuclei. The g principal values are g1 = 1.9670, g2 = 2.1122, and g3=2.0043, with the g1 and g2 axes pointing at the nitrogens of the corrole macrocycle and the g3 axis directed perpendicular to the plane. The cobalt hyperfine...

Hyperfine selectivity using multiquantum electron–nuclear–electron triple resonance

Hyperfine selectivity is demonstrated in a continuous wave electron–nuclear double resonance (ENDOR) experiment. A multiquantum electron–electron double resonance (ELDOR) signal is monitored as a function of the nuclear radio frequency. The signs and relative intensities of the ENDOR lines permit separating the case where both ELDOR and ENDOR frequencies match hyperfine couplings from the cases where this condition is not satisfied.