Multi-frequency EPR and Mössbauer spectroscopic studies on freeze-quenched reaction intermediates of nitric oxide synthase.

It is believed by analogy to chloroperoxidase (CPO) from Caldariomyces fumago that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of nitric oxide synthase (NOS) corresponds to an iron(IV) porphyrin-pi -cation radical. Such species can also be produced by the reaction of ferric NOS with external oxidants within the shunt pathway. We present multi-frequency EPR (9.6, 94, 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates of the oxygenase domain of nitric oxide synthase which has reacted with peroxy acetic acid within 8-200 ms. The intermediates of the oxygenase domain of both the cytokine inducible NOS (iNOSox) and the neuronal NOS (nNOSox) show an organic radical signal in the 9.6-GHz spectrum overlapping with the spectrum of an unknown species with g-values of 2.24, 2.23 and 1.96. Using 94- and 285-GHz EPR the organic radical signal is assigned to a tyrosine radical on the basis of g-values (i.e. Tyr*562 in nNOSox and Tyr*341 in iNOSox). Mössbauer spectroscopy of (57)Fe-labeled unreacted nNOSox shows a ferric low-spin heme-iron (delta = 0.38 mms(-1), deltaE(Q) = 2.58 mms(-1)). The reaction of nNOSox with peroxy acetic acid for 8 ms leads to the disappearance of the magnetic background characteristic for native nNOSox and a new species with delta = 0.27 mms(-1) and deltaE(Q) = 2.41 mms(-1) is detected at 4.2 K which does not resemble the parameters typical for a Fe(IV) center. It is proposed that this intermediate species corresponds to a ferric low-spin species which magnetically couples to an amino acid radical (presumably Trp*409).

A stable FeIII-FeIV replacement of tyrosyl radical in a class I ribonucleotide reductase.

Ribonucleotide reductase (RNR) of Chlamydia trachomatis is a class I RNR enzyme composed of two homodimeric components, proteins R1 and R2. In class I RNR, R1 has the substrate binding site, whereas R2 has a diferric site and normally in its active form a stable tyrosyl free radical. C. trachomatis RNR is unusual, because its R2 component has a phenylalanine in the place of the radical carrier tyrosine. Replacing the tyrosyl radical, a paramagnetic Fe(III)-Fe(IV) species (species X, normally a transient intermediate in the process leading to radical formation) may provide the oxidation equivalent needed to start the catalytic process via long range electron transfer from the active site in R1. Here EPR spectroscopy shows that in C. trachomatis RNR, species X can become essentially stable when formed in a complete RNR (R1/R2/substrate) complex, adding further weight to the possible role of this species X in the catalytic reaction.

Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz.

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: the tyrosine radical Y(D)(*).

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.

Single crystal EPR studies of the oxidized active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

The Ni-A and the Ni-B forms of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F have been studied in single crystals by continuous wave and pulsed EPR spectroscopy at different temperatures (280 K, 80 K, and 10 K). For the first time, the orientation of the g-tensor axes with respect to the recently published atomic structure of the active site at 1.8 A resolution was elucidated for Ni-A and Ni-B. The determined g-tensors have a similar orientation. The configuration of the electronic ground state is proposed to be Ni(III) 3d1/z 2 for Ni-A and Ni-B. The gz principal axis is close to the Ni-S(Cys549) direction; the gx and the gy axes are approximately along the Ni-S(Cys546) and Ni-S(Cys81) bonds, respectively. It is proposed that the difference between the Ni-A and Ni-B states lies in a protonation of the bridging ligand between the Ni and the Fe.

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.

Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides.

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll-protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to approximately 60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron-nuclear-nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from approximately 2:1 to approximately 5:1 in the mutants compared with approximately 2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation-reduction midpoint potential.

Electronic structure of Q-A in reaction centers from Rhodobacter sphaeroides. I. Electron paramagnetic resonance in single crystals.

The magnitude and orientation of the electronic g-tensor of the primary electron acceptor quinone radical anion, Q-A, has been determined in single crystals of zinc-substituted reaction centers of Rhodobacter sphaeroides R-26 at 275 K and at 80 K. To obtain high spectral resolution, EPR experiments were performed at 35 GHz and the native ubiquinone-10 (UQ10) in the reaction center was replaced by fully deuterated UQ10. The principal values and the direction cosines of the g-tensor axes with respect to the crystal axes a, b, c were determined. Freezing of the single crystals resulted in only minor changes in magnitude and orientation of the g-tensor. The orientation of Q-A as determined by the g-tensor axes deviates only by a few degrees (< or = 8 degrees) from the orientation of the neutral QA obtained from an average of four different x-ray structures of Rb. sphaeroides reaction centers. This deviation lies within the accuracy of the x-ray structure determinations. The g-tensor values measured in single crystals agree well with those in frozen solutions. Variations in g-values between Q-A, Q-B, and UQ10 radical ion in frozen solutions were observed and attributed to different environments.

ENDOR studies of the primary donor cation radical in mutant reaction centers of Rhodobacter sphaeroides with altered hydrogen-bond interactions.

The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides. The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll dimer, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His-->Phe, HF(L168), and M197 Phe-->His, FH(M197), near the 2-acetyl groups of the dimer and at sites M160 Leu-->His, LH(M160), and L131 Leu-->His, LH(L131), in the vicinity of the 9-keto carbonyls of the dimer. The single mutants and a complete set of double mutants were studied using EPR, ENDOR, and TRIPLE resonance spectroscopy. The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the dimer oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Williams, J. C. & Allen, J. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269]. It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the dimer. Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the dimer to a localization on the M-half, depending on the hydrogen bond situation. As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M202), which contains a BChlL-BPheM heterodimer as the primary donor with localized spin on the BChl aL [Bylina, E. J., & Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7226-7230; Schenck, C. C., Gaul, D., Steffen M., Boxer S. G., McDowell L., Kirmaier C., & Holten D. (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M. E., Ed.) pp 229-238, Springer, Berlin] were studied. In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed. The effects of the introduced hydrogen bonds on the spin density distribution of the dimer in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation. The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

EPR and ENDOR investigation of the primary electron acceptor radical anion QA.- in iron-depleted photosystem II membrane fragments.

Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones QA and QB in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9.- in 2-propanol and QA.- in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9.- in vitro and on QA.- in the iron-depleted PS II samples. For the former a complete set of isotropic 1H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. 1H-ENDOR spectra of QA.- in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of QA and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.

Comparative study of reaction centers from photosynthetic purple bacteria: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Reaction centers (RCs) from four species of purple bacteria, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and the recently discovered bacterium Rhodospirillum centenum, have been characterized by optical spectroscopy [Wang, S., Lin, X., Woodbury, N. W., & Allen, J. P. (1994) Photosynth. Res. (submitted for publication)] and magnetic resonance spectroscopy. All RCs contain a bacteriochlorophyll (BChl) a dimer as the primary donor. For Rb. sphaeroides and Rs. rubrum the donor QY optical band is at approximately 865 nm, compared to approximately 850 nm for Rb. capsulatus and Rs. centenum. The primary donor in the RCs can be converted between these two forms by the addition or removal of charged detergents. The electronic structure of the cation radical of the primary electron donor P+. was investigated in these species using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and electron nuclear triple resonance (TRIPLE) spectroscopy. The EPR line widths of P+. vary significantly and the ENDOR and Special TRIPLE spectra reveal drastic differences in the spin density distribution of the dimer for the different species. Reaction centers from Rb. sphaeroides and Rs. rubrum have a slightly asymmetric spin density distribution over the two halves of the dimer. The respective ratios are 2:1 and 1.6:1 in favor of the L-half of the BChl a dimer. In contrast, the spectra of P+. in reaction centers from Rb. capsulatus and Rs. centenum show an almost complete localization of the unpaired electron on the L-half of the dimer (ratio approximately 5:1).(ABSTRACT TRUNCATED AT 250 WORDS)

Structural studies of the primary donor cation radical P(870) in reaction centers of Rhodospirillum rubrum by electron-nuclear double resonance in solution.

The light-induced cation radical of the primary electron donor, P(870) (+.), in photosynthetic reaction centers from Rhodospirillum rubrum G-9, has been investigated by electron-nuclear double resonance (ENDOR) in liquid aqueous solution. The measured hyperfine coupling constants are assigned to specific molecular positions by partial deuteration. Comparison with the bacteriochlorophyll a cation radical shows different reduction factors of the individual coupling constants deviating from the value 2.0 reported in earlier investigations in frozen solutions. The average of the coupling constants is, however, reduced by a factor very close to 2.0. EPR simulations using the ENDOR coupling constants support a dimer model for P(870) (+.) with C(2) symmetry, where the two macrocycles are close enough to form a supermolecular orbital resulting in a different distribution of the unpaired electron, compared with the monomeric bacteriochlorophyll a cation radical. Molecular orbital calculations were used to obtain structural information about this dimer.