R.A. Isaacson

Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides

The photoreductive trapping of the transient, intermediate acceptor, I-, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I- was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 400, 587--599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301--304). The optical changes of I- showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I- were observed: one was a narrow singlet at g = 2.0035, deltaH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I- on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI-Q-Fe2+ and the singlet EPR signal is due to DI-Q2-Fe2+. : formula: (see text), where D is the primary donor (BChl)2+. The above model was supported by the following observations: (1) During the first illumination, sigmoidal kinetics of the formation of I- was observed. This is a direct consequence of the three-sequential reactions. (2) During the second and subsequent illuminations first-order (exponential) kinetics were observed for the formation of I-. This is due to the dark decay, k4, to the state DIQ2-Fe2+ formed after the first illumination. (3) Removal of the quinone resulted in first-order kinetics. In this case, only the first step, k1, is operative. (4) The observation of the doublet signal in reaction centers containing menaquinone but not ubiquinone is explained by the longer lifetime of the doublet species I-(Q-Fe2%) in reaction centers containing menaquinone. The value of tau2 was determined from kinetic measurements to be 0.01 s for ubiquinone and 4 s for menaquinone (T = 20 degrees C). The temperature and pH dependence of the dark electron transfer reaction I-(Q-Fe2+) yields I(Q2-Fe2+) was studied in detail. The activation energy for this process was found to be 0.42 eV for reaction centers containing ubiquinone and 0.67 eV for reaction centers with menaquinone. The activation energy and the doublet splitting were used to calculate the rate of electron transfer from I- to MQ-Fe2+ using Hopfields theory for thermally activated electron tunneling. The calculated rate agrees well with the experimentally determined rate which provides support for electron tunneling as the mechanism for electron transfer in this reaction. Using the EPR doublet splitting and the activation energy for electron transfer, the tunneling matrix element was calculated to be 10(-3) eV. From this value the distance between I- and MQ- was estimated to be 7.5--10 A.

The electronic structure of the primary donor cation radical in Rhodobacter sphaeroides R-26: ENDOR and TRIPLE resonance studies in single crystals of reaction centers

Abstract The electron spin density distribution of the cation radical of the primary donor, D+, a bacteriochlorophyll a dimer was determined by ENDOR and TRIPLE resonance experiments performed on single crystals of reaction centers (RCs) of Rhodobacter sphaeroides R-26. Nine isotropic proton hyperfine coupling constants (hfcs) were obtained and from the angular dependence of the hfcs in three crystallographic planes, five complete hyperfine (hf) tensors were determined. Theoretical hf tensors were calculated by the all-valence-electron SCF molecular orbital method RHF-INDO/SP using the X-ray structure data of the dimer D and its amino acid environment. A comparison of the directions of the principal axes of the experimental and calculated hf tensors enabled us to identify the hfcs with specific protons on the two bacteriochlorophyll halves DL and DM of the dimer. The result shows that the unpaired valence electron is unequally distributed over the dimer halves, favoring DL by approx. 2:1. This ratio has been obtained from the proton hfcs of rotating methyl groups, which directly reflect the π-spin densities at the corresponding positions in the two macrocycles, DL and DM. It was further confirmed by recent 15N-ENDOR experiments on RC single crystals (Lendzian, F., Bonigk, B., Plato, M., Mobius, K. and Lubitz, W. (1992) in The Photosynthetic Bacterial Reaction Center II (Breton, J. and Vermeglio, A., eds.), pp. 89–97, Plenum Press, New York). The observed asymmetry of D+ is attributed to the difference in energies of the highest filled molecular π-orbitals of the monomeric halves, DL and DM, which is caused by differences in the structure of the two bacteriochlorophylls and/or their environment. Possible implications of this asymmetry for the electron transfer in the RC are discussed.

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex

Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.

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.

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.

On the question of the primary acceptor in bacterial photosynthesis: Manganese substituting for iron in reaction centers of Rhodopseudomonas spheroides R-26

Abstract Iron was partially replaced by manganese in a reaction center preparation of Rhodopseudomonas spheroides R-26. The reaction centers containing manganese were distinguished spectroscopically (EPR) from those containing iron. The low-temperature photochemical activities were found to be identical for both species. This makes it unlikely that the transition metal by itself is the primary electron acceptor.

Probing hydrogen bonding to quinone anion radicals by 1H and 2H ENDOR spectroscopy at 35 GHz

Abstract ENDOR spectroscopy at 35 GHz and 80 K was used to study the radical anion of 1,4-benzoquinone (BQ) in water and various alcohols. BQ -d 4 in H 2 O and BQ -h 4 in D 2 O were investigated with the aim to obtain information on the hydrogen bonds between the quinone oxygen and the respective solvent. The observed spectra were analyzed using the GENDOR program for the simulation of orientationally selected powder ENDOR spectra. From the spectral simulations the hyperfine coupling and nuclear quadrupolar coupling tensor components and the angles with respect to the quinone axes were obtained. Hydrogen bond lengths and geometries were deduced from the hyperfine couplings using the point-dipole model and from the nuclear quadrupolar couplings using empirical relationships. The experimental results were compared, and found to be in fair agreement, with those derived from Density Functional Theory (DFT) calculations performed on geometry optimized structures of solute and solvent.

Determination of the zero-field splitting of Fe3+ in heme proteins from the temperature dependence of the spin-lattice relaxation rate

Abstract A sensitive method for obtaining the zero-field crystalline field splitting is described and applied to ferrimyoglobin (H 2 O) and ferrimyoglobin (F − ). The high accuracy (1–2%) with which the splitting can be determined should make it possible to explore the environmental (conformatinal) changes at the Fe 3+ site.

A tunable general purpose Q-band resonator for CW and pulse EPR/ENDOR experiments with large sample access and optical excitation.

We describe a frequency tunable Q-band cavity (34 GHz) designed for CW and pulse Electron Paramagnetic Resonance (EPR) as well as Electron Nuclear Double Resonance (ENDOR) and Electron Electron Double Resonance (ELDOR) experiments. The TE(011) cylindrical resonator is machined either from brass or from graphite (which is subsequently gold plated), to improve the penetration of the 100 kHz field modulation signal. The (self-supporting) ENDOR coil consists of four 0.8mm silver posts at 2.67 mm distance from the cavity center axis, penetrating through the plunger heads. It is very robust and immune to mechanical vibrations. The coil is electrically shielded to enable CW ENDOR experiments with high RF power (500 W). The top plunger of the cavity is movable and allows a frequency tuning of ±2 GHz. In our setup the standard operation frequency is 34.0 GHz. The microwaves are coupled into the resonator through an iris in the cylinder wall and matching is accomplished by a sliding short in the coupling waveguide. Optical excitation of the sample is enabled through slits in the cavity wall (transmission ∼60%). The resonator accepts 3mm o.d. sample tubes. This leads to a favorable sensitivity especially for pulse EPR experiments of low concentration biological samples. The probehead dimensions are compatible with that of Bruker flexline Q-band resonators and it fits perfectly into an Oxford CF935 Helium flow cryostat (4-300 K). It is demonstrated that, due to the relatively large active sample volume (20-30 μl), the described resonator has superior concentration sensitivity as compared to commercial pulse Q-band resonators. The quality factor (Q(L)) of the resonator can be varied between 2600 (critical coupling) and 1300 (over-coupling). The shortest achieved π/2-pulse durations are 20 ns using a 3 W microwave amplifier. ENDOR (RF) π-pulses of 20 μs ((1)H @ 51 MHz) were obtained for a 300 W amplifier and 7 μs using a 2500 W amplifier. Selected applications of the resonator are presented.

ELECTRON NUCLEAR DOUBLE RESONANCE (ENDOR) INVESTIGATION ON MYOGLOBIN AND HEMOGLOBIN

Heme proteins play a vital role in many biological processes: the storage of oxygen (myoglobin) , the transport of oxygen (hemoglobin), the shuttling of electrons (cytochromes) and enzymatic reactions (e.g., peroxidases) , to name a few. Many different techniques have been used in the past to elucidate the structure of heme proteins. These include x-ray diffraction, optical spectroscopy, Mossbauer spectroscopy, high resolution nuclear magnetic resonance (nmr) and electron paramagnetic resonance (epr) spectroscopy. Within the past year we have started a program to investigate heme proteins by the electron nuclear double resonance (endor) technique. In this paper we present some results on myoglobin and hemoglobin. Some of these are still preliminary in nature and should therefore be taken in the spirit of a progress report. What can endor do that hasn’t been already done by the other techniques? It can determine, with a high degree of precision, the interactions of the paramagnetic ion, in our case the iron (Fe+++) of the heme, with its surrounding nuclei. From the value of these interactions one may obtain the electron cloud distribution (i.e., the square of the electronic wave functions) at the nuclei, the distance of the iron to the nuclei and the electric field gradient at the nuclei, in short, the intimate electronic details of the iron and its environment. Since the iron represents the “business end” of the molecule, such detailed information should lead to a more complete, quantum mechanical understanding of the relationship between electronic structure and function. Changes in the structure or in the conformation of the heme and its environment are also expected to show up in a change in the endor spectrum. A problem of central interest in which such changes play a major role is the cooperative oxygenation effect in hemoglobin.