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The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.
Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).
One of the central research topics in the field of biophysical chemistry is the structure and function of membrane proteins involved in energy transduction. Both, the aerobic and the anaerobic respiration include electron transfer and proton translocation across the mitochondrial and bacterial membranes. These electron transfer processes lead to changes in oxidation states of cofactors some of which are paramagnetic. Therefore, EPR spectroscopy is the method of choice to obtain electronic and structural information directly related to the function of the respiratory chain proteins. In this work, multifrequency continuous wave (CW) and pulsed EPR spectroscopy has been used to characterize the molybdenum active site of polysulfide reductase (Psr) from the anaerobic bacterium Wolinella succinogenes and the protein-protein complex between cytochrome c oxidase (CcO) and cytochrome c from the aerobic bacterium Paracoccus denitrificans. Molybdenum in Psr-Psr is an enzyme essential for the sulfur respiration of Wolinella succinogenes. Biochemical studies suggested that the active site of this enzyme contains a mononuclear Mo center, which catalyzes the reduction of the substrate polysulfide to sulfide. Until now there is no crystal structure available for Psr. Consequently, current characterizations of this enzyme have to rely on biochemical and spectroscopic investigations. Within the present work, CW and modern pulsed EPR techniques were applied to investigate its catalytically active site. In the first part of this thesis, different redox agents have been used to generate paramagnetic states of Psr. Multifrequency CW-EPR spectroscopy was applied to identify the Mo(V) states. Using simulations of the experimental spectra, three spectroscopically distinct states have been identified based on the Mo hyperfine- and g-tensor values. Comparison of their EPR parameters with those of related enzymes indicated five or six sulfur ligands at the Mo center depending on the state. The state generated by addition of polysulfide is suggested to be the catalytically active form, in which the Mo is coordinated by a sulfur of the polysulfide chain as the sixth ligand. 33S (I = 3/2) labeled polysulfide was prepared to probe the proximity of the polysulfide to the molybdenum center via its hyperfine coupling. 1D-ESEEM and 2D122 HYSCORE spectroscopy was used to detect these hyperfine and quadrupole interactions, which are too small to be observed in conventional CW EPR spectra. To date there has been only one pulsed-EPR study involving a 33S nucleus [Finazzo et.al. 2003]. The reasons are that this nucleus has a high nuclear spin of I = 3/2 and a large nuclear quadrupole moment in addition to the low Larmor frequency. All these make the detection of sulfur and the extraction of structural information demanding. However, analysis of the 2D-data led to a Mo(V) 33S distance in a range of about 2 to 2.5 Å. Mo-S distances found in molybdenum enzymes of the same family are in a range of 1.8 to 2.8 Å suggesting that the 33S is indeed the sixth ligand of the Mo(V) center and demonstrating that polysulfide is the actual substrate for this enzyme. Thus HYSCORE experiments have been proved to be a powerful technique to gain further insight into the active site structures of molybdenum enzymes and the trafficking of substrate atoms during catalysis. Density functional theory (DFT) calculations together with quantitative numerical simulations of the 2D-data will help to obtain more structural details about the molybdenum binding site in Psr. CcO:cytochrome c complex Protein-protein complex formation is an important step in energy conversion biological processes such as respiration and photosynthesis. These protein-protein complexes are involved in long range electron transfer reactions and are known to be of transient nature. Within the bacterial and mitochondrial respiratory electron transport chains such a complex is formed between CcO and cytochrome c. Upon complex formation cytochrome c donates the electrons required for the CcO catalyzed reduction of dioxygen to water. Here, the protein-protein complex formation between CcO and cytochrome c from Paracoccus denitrificans was investigated by pulsed EPR spectroscopy. The idea was to use the relaxation enhancement due to the distance and orientation dependent magnetic dipole-dipole interaction between the paramagnetic centers in the different CcO constructs and cytochromes. Two-pulse electron spin echo experiments were carried out on mixtures of the CuA containing soluble subunit II or the full size CcO with the physiological partner cytochrome c552 or horse heart cytochrome c. Significantly enhanced relaxation of CuA due to specific protein-protein complex formation has been observed in all four cases. In contrast the non-binding cytochrome c1 showed only a very weak relaxation enhancement due to unspecific protein-protein interactions. The echo decays of the slowly relaxing observer spin (CuA of CcO) measured in the absence and presence of the fast relaxing spin (Fe(III) of cytochrome c) permitted the extraction of the pure dipolar relaxation contributions for the different complexes. Measurements at different temperatures proved the dipolar nature of the relaxation enhancement. Furthermore, it was demonstrated experimentally that this approach also works for the full-size CcO, which contains four paramagnetic metal centers, in complex with cytochrome c. Quantitative simulations of the data suggest a broad distribution in distances (2 - 4 nm) and orientations between the CuA and Fe(III) in the complex between CcO and cytochrome c. High-field EPR spectroscopy will be useful to further analyze and prove these complex structures. Within the present work, it has been shown that pulsed relaxation enhancement experiments can be used to investigate the distance and relative orientation between paramagnetic metal centers. Furthermore, it has been demonstrated on a qualitative level, that this method can be used complimentary to other biophysical approaches to study transient electron transfer protein-protein complexes. Finally, within this work it has been proven that this method can be applied also to biological systems where more than two paramagnetic centers are present. This is particularly interesting for supercomplexes between membrane proteins.