Model for magnetic field effects on radical pair recombination in enzyme kinetics.

A prototypical model for describing magnetic field effects on the reaction kinetics of enzymes that exhibit radical pair recombination steps in their reaction cycle is presented. The model is an extended Michaelis-Menten reaction scheme including an intermediate enzyme-substrate complex where a spin-correlated radical pair state exists. The simple structure of the scheme makes it possible to calculate the enzyme reaction rate explicitly by combining chemical kinetics with magnetic field-dependent spin kinetics (radical pair mechanism). Recombination probability is determined by using the exponential model. Simulations show that the size of the magnetic field effect depends on relations between different rate constants, such as 1) the ratio between radical pair-lifetime and the magnetic field-sensitive intersystem crossing induced by the hyperfine interaction and the delta g mechanisms and 2) the chemical rate constants of the enzyme reaction cycle. An amplification factor that is derived from the specific relations between the rate constants is defined. It accounts for the fact that although the magnetic field-induced change in radical pair recombination probability is very small, the effect on the enzyme reaction rate is considerably larger, for example, by a factor of 1 to 100. Model simulations enable a qualitative comparison with recent experimental studies reporting magnetic field effects on coenzyme B12-dependent ethanolamine ammonia lyase in vitro activity that revealed a reduction in Vmax/KM at low flux densities and a return to the zero-field rate or an increase at high flux densities.     

The Influence of a gradient static magnetic field on an unstirred Belousov-Zhabotinsky reaction

It is believed that static magnetic fields (SMF) cannot affect the pattern formation of the Belousov-Zhabotinsky (BZ) reaction, which has been frequently studied as a simplified experimental model of a nonequilibrium open system, because SMF produces no induced current and the magnetic force of SMF far below 1 T is too low to expect the effects on electrons in the BZ reaction. In the present study, we examined whether the velocity of chemical waves in the unstirred BZ reaction can be affected by a moderate-intensity SMF exposure depending on the spatial magnetic gradient. The SMF was generated by a parallel pair of attracting rectangular NdFeB magnets positioned opposite each other. The respective maximum values of magnetic flux density (B(max)), magnetic flux gradient (G(max)), and the magnetic force product of the magnetic flux density its gradient (a magnetic force parameter) were 206 mT, 37 mT/mm, and 3,000 mT(2)/mm. The ferroin-catalyzed BZ medium was exposed to the SMF for up to 16 min at 25 degrees C. The experiments demonstrated that the wave velocity was significantly accelerated primarily by the magnetic gradient. The propagation of the fastest wave front indicated a sigmoid increase along the peak magnetic gradient line, but not along the peak magnetic force product line. The underlying mechanisms of the SMF effects on the anomalous wave propagation could be attributed primarily to the increased concentration gradient of the paramagnetic iron ion complexes at the chemical wave fronts induced by the magnetic gradient.     

Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D⁺ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone-iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.     

Growth of human cultured cells exposed to a non-homogeneous static magnetic field generated by Sm-Co magnets.

A static magnetic field, with a strong spatial gradient, was established on the surface of cell culture dishes by use of a gilded iron needle set vertically above an Sm-Co magnet. The calculated magnetic flux density was more than 1.5 T at the center of the needle tip, and the products of the flux density and its gradient were about 200 and 60 T2/m at distances of 0.1 and 0.3 mm, respectively, from the center. The DNA content, DNA synthesis and labeling index of cultured cells located within 0.1 mm from the center of the needle, and the growth rate of cells located within 0.3 mm from the center, were measured. HeLa cells grew at a normal rate for 96 h in the magnetic field and showed no significant change in shape, detectable by scanning electron microscopy. The growth of HeLa cells was not influenced by exposure to the magnetic field. Similarly, exposure for 48 h to the magnetic field had no effect on growth of normal human gingival fibroblasts (Gin-1). The DNA content, assayed by microfluorometry of the nuclei of both types of cells stained by the Feulgen reaction, was not significantly different from that of controls. Moreover, exposure to the magnetic field had no effect on DNA synthesis or the labeling index of HeLa cells assayed by autoradiography of incorporated [3H]thymidine. It is concluded that a non-homogeneous magnetic field of the intensity and the gradient used in this study does not significantly influence the growth of HeLa cells or Gin-1 cells.     

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.     

Low-temperature pulsed EPR study at 34 GHz of the triplet states of the primary electron Donor P865 and the carotenoid in native and mutant bacterial reaction centers of Rhodobacter sphaeroides

The photosynthetic charge separation in bacterial reaction centers occurs predominantly along one of two nearly symmetric branches of cofactors. Low-temperature EPR spectra of the triplet states of the chlorophyll and carotenoid pigments in the reaction center of Rhodobacter sphaeroides R-26.1, 2.4.1 and two double-mutants GD(M203)/AW(M260) and LH(M214)/AW(M260) have been recorded at 34 GHz to investigate the relative activities of the "A" and "B" branches. The triplet states are found to derive from radical pair and intersystem crossing mechanisms, and the rates of formation are anisotropic. The former mechanism is operative for Rb. sphaeroides R-26.1, 2.4.1, and mutant GD(M203)/AW(M260) and indicates that A-branch charge separation proceeds at temperatures down to 10 K. The latter mechanism, derived from the spin polarization and operative for mutant LH(M214)/AW(M260), indicates that no long-lived radical pairs are formed upon direct excitation of the primary donor and that virtually no charge separation at the B-branch occurs at low temperatures. When the temperature is raised above 30 K, B-branch charge separation is observed, which is at most 1% of A-branch charge separation. B-branch radical pair formation can be induced at 10 K with low yield by direct excitation of the bacteriopheophytin of the B-branch at 590 nm. The formation of a carotenoid triplet state is observed. The rate of formation depends on the orientation of the reaction center in the magnetic field and is caused by a magnetic field dependence of the oscillation frequency by which the singlet and triplet radical pair precursor states interchange. Combination of these findings with literature data provides strong evidence that the thermally activated transfer step on the B-branch occurs between the primary donor, P865, and the accessory bacteriochlorophyll, whereas this step is barrierless down to 10 K along the A-branch.     

Origin of the transient electron paramagnetic resonance signals in DNA photolyase

DNA photolyase repairs pyrimidine dimer lesions in DNA through light-induced electron donation to the dimer. During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron-oxidized to a semiquinone radical. In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue. This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR. By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin. The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan. We also report a new procedure for the rapid isolation of DNA photolyase.     

Effect of static magnetic fields on the budding of yeast cells

The effect of static magnetic fields on the budding of single yeast cells was investigated using a magnetic circuit that was capable of generating a strong magnetic field (2.93 T) and gradient (6100 T² m^?1). Saccharomyces cerevisiae yeast cells were grown in an aqueous YPD agar in a silica capillary under either a homogeneous or inhomogeneous static magnetic field. Although the size of budding yeast cells was only slightly affected by the magnetic fields after 4 h, the budding angle was clearly affected by the direction of the homogeneous and inhomogeneous magnetic fields. In the homogeneous magnetic field, the budding direction of daughter yeast cells was mainly oriented in the direction of magnetic field B. However, when subjected to the inhomogeneous magnetic field, the daughter yeast cells tended to bud along the axis of capillary flow in regions where the magnetic gradient, estimated by B(dB/dx), were high. Based on the present experimental results, the possible mechanism for the magnetic effect on the budding direction of daughter yeast cells is theoretically discussed. Bioelectromagnetics 31:622–629, 2010. © 2010 Wiley‐Liss, Inc.     

Transient radical pairs studied by time-resolved EPR

Photogenerated short-lived radical pairs (RP) are common in biological photoprocesses such as photosynthesis and enzymatic DNA repair. They can be favorably probed by time-resolved electron paramagnetic resonance (EPR) methods with adequate time resolution. Two EPR techniques have proven to be particularly useful to extract information on the working states of photoinduced biological processes that is only difficult or sometimes even impossible to obtain by other types of spectroscopy. Firstly, transient EPR yields crucial information on the chemical nature and the geometry of the individual RP halves in a doublet-spin pair generated by a short laser pulse. This time-resolved method is applicable in all magnetic field/microwave frequency regimes that are used for continuous-wave EPR, and is nowadays routinely utilized with a time resolution reaching about 10 ns. Secondly, a pulsed EPR method named out-of-phase electron spin echo envelope modulation (OOP-ESEEM) is increasingly becoming popular. By this pulsed technique, the mutual spin-spin interaction between the RP halves in a doublet-spin pair manifests itself as an echo modulation detected as a function of the microwave-pulse spacing of a two-pulse echo sequence subsequent to a laser pulse. From the dipolar coupling, the distance between the radicals is readily derived. Since the spin-spin interaction parameters are typically not observable by transient EPR, the two techniques complement each other favorably. Both EPR methods have recently been applied to a variety of light-induced RPs in photobiology. This review summarizes the results obtained from such studies in the fields of plant and bacterial photosynthesis and DNA repair mediated by the enzyme DNA photolyase.     

Transient radical pairs studied by time-resolved EPR

Photogenerated short-lived radical pairs (RP) are common in biological photoprocesses such as photosynthesis and enzymatic DNA repair. They can be favorably probed by time-resolved electron paramagnetic resonance (EPR) methods with adequate time resolution. Two EPR techniques have proven to be particularly useful to extract information on the working states of photoinduced biological processes that is only difficult or sometimes even impossible to obtain by other types of spectroscopy. Firstly, transient EPR yields crucial information on the chemical nature and the geometry of the individual RP halves in a doublet-spin pair generated by a short laser pulse. This time-resolved method is applicable in all magnetic field/microwave frequency regimes that are used for continuous-wave EPR, and is nowadays routinely utilized with a time resolution reaching about 10 ns. Secondly, a pulsed EPR method named out-of-phase electron spin echo envelope modulation (OOP-ESEEM) is increasingly becoming popular. By this pulsed technique, the mutual spin-spin interaction between the RP halves in a doublet-spin pair manifests itself as an echo modulation detected as a function of the microwave-pulse spacing of a two-pulse echo sequence subsequent to a laser pulse. From the dipolar coupling, the distance between the radicals is readily derived. Since the spin-spin interaction parameters are typically not observable by transient EPR, the two techniques complement each other favorably. Both EPR methods have recently been applied to a variety of light-induced RPs in photobiology. This review summarizes the results obtained from such studies in the fields of plant and bacterial photosynthesis and DNA repair mediated by the enzyme DNA photolyase.     

Modulation of the shape and speed of a chemical wave in an unstirred Belousov–Zhabotinsky reaction by a rotating magnet

The objective of this study was to observe whether a rotating magnetic field (RMF) could change the anomalous chemical wave propagation induced by a moderate‐intensity gradient static magnetic field (SMF) in an unstirred Belousov–Zhabotinsky (BZ) reaction. The application of the SMF (maximum magnetic flux density = 0.22 T, maximum magnetic flux density gradient = 25.5 T/m, and peak magnetic force product (flux density × gradient) = 4 T²/m) accelerated the propagation velocity in a two‐dimensional pattern. Characteristic anomalous patterns of the wavefront shape were generated and the patterns were dependent on the SMF distribution. The deformation and increase in the propagation velocity were diminished by the application of an RMF at a rotation rate of 1 rpm for a few minutes. Numerical simulation by means of the time‐averaged value of the magnetic flux density gradient or the MF gradient force over one rotation partially supported the experimental observations. These considerations suggest that RMF exposure modulates the chemical wave propagation and that the degree of modulation could be, at least in part, dependent on the time‐averaged MF distribution over one rotation. Bioelectromagnetics 34:220–230, 2013. © 2012 Wiley Periodicals, Inc.     

The cytochrome c oxidase-azide-nitric oxide complex as a model for the oxygen-binding site

The complex of cytochrome c oxidase with NO and azide has been studied by EPR at 9.2 and 35 GHz. This complex which shows delta ms = 2 EPR triplet and strong anisotropic signals, due to the interaction of cytochrome a2+3 X NO (S = 1/2) and Cu2+B (S = 1/2), is photodissociable . Its action spectrum is similar to that of cytochrome a2+3 X NO with bands at 430, 560 and 595 nm, but shows an additional band in the near ultraviolet region. The quantum yield of the photodissociation process of cytochrome a2+3 X NO in the metal pair appears to depend on the redox state of CuB. When the photolysed sample was warmed to 77 K, a complex was observed with the EPR parameters of cytochrome a3+3 - N-3 - Cu1 +B (S = 1/2). This process of electron and ligand transfer can be reversed by heating the sample to 220 K. It is suggested that in the triplet species azide is bound to Cu2+B whereas NO is bridged between Cu2+B and the haem iron of the cytochrome a2+3. The complex has a triplet ground state and a singlet excited state with an exchange interaction J = -7.1 cm-1 between both spins. The anisotropy in the EPR spectra is mainly due to a magnetic dipole-dipole interaction between cytochrome a2+3 X NO and Cu2+B. From simulations of the triplet EPR spectra obtained at 9 and 35 GHz, a value for the distance between the nitroxide radical and Cu2+B of 0.33 nm was found. A model of the NO binding in the cytochrome a3-Cu pair shows a distance between the haem iron of cytochrome a3 and CuB of 0.45 nm. It is concluded that the cytochrome a3-CuB pair forms a cage in which the dioxygen molecule is bidentate coordinated to the two metals during the catalytic reaction.     

Magnetic studies of the four-iron high-potential, non-heme protein from Chromatium vinosum.

Extensive EPR studies on high-potential, iron-sulfur protein from Chromatium vinosum indicate that the singular spectrum of this four-iron, non-heme protein consists of a superposition of three distinct signals; namely, two principal signals of equal weight, one reflecting axial and the other rhombic symmetry, and a third nearly isotropic minority component. In addition, magnetic susceptibility experiments on two oxidation states of the protein from 4.2 to approx. 260 degrees K indicate antiferromagnetic exchange coupling between iron atoms. Possible origins of the complex EPR signals are discussed, and a preferred model that is consistent with EPR, magnetic susceptibility, NMR, X-ray, and M?ssbauer data is presented.     

Split EPR signals from photosystem II are modified by methanol, reflecting S state-dependent binding and alterations in the magnetic coupling in the CaMn4 cluster

Methanol binds to the CaMn4 cluster in photosystem II (PSII). Here we report the methanol dependence of the split EPR signals originating from the magnetic interaction between the CaMn4 cluster and the Y(Z)* radical in PSII which are induced by illumination at 5 K. We found that the magnitudes of the "split S1" and "split S3" signals induced in the S1 and S3 states of PSII centers, respectively, are diminished with an increase in the methanol concentration. The methanol concentrations at which half of the respective spectral changes had occurred ([MeOH](1/2)) were 0.12 and 0.57%, respectively. By contrast, the "split S0" signal induced in the S0 state is broadened, and its amplitude is enhanced. [MeOH](1/2) for this change was found to be 0.54%. We discuss these observations with respect to the location and nature of the methanol binding site. Furthermore, by comparing this behavior with methanol effects reported for other EPR signals in the different S states, we propose that the observed methanol-dependent changes in the split S1 and split S0 EPR signals are caused by an increase in the extent of magnetic coupling within the cluster.     

Heme spin states and peroxide-induced radical species in prostaglandin H synthase

We have examined the optical, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectra of pure ovine prostaglandin H synthase in its resting (ferric) and ferrous states and after addition of hydrogen peroxide or 15-hydroperoxyeicosatetraenoic acid. In resting synthase, the distribution of heme between high- and low-spin forms was temperature-dependent: 20% of the heme was low-spin at room temperature whereas 50% was low-spin at 12 K. Two histidine residues were coordinated to the heme iron in the low-spin species. Anaerobic reduction of the synthase with dithionite produced a high-spin ferrous species that had no EPR signals. Upon reaction with the resting synthase, both hydroperoxides quickly generated intense (20-40% of the synthase heme) and complex EPR signals around g = 2 that were accompanied by corresponding decreases in the intensity of the signals from ferric heme at g = 3 and g = 6. The signal generated by HOOH had a doublet at g = 2.003, split by 22 G, superimposed on a broad component with a peak at g = 2.085 and a trough at g = 1.95. The lipid hydroperoxide generated a singlet at g = 2.003, with a linewidth of 25 G, superimposed on a broad background with a peak at g = 2.095 and a trough around g = 1.9. These EPR signals induced by hydroperoxide may reflect synthase heme in the ferryl state complexed with a free radical derived from hydroperoxide or fragments of hydroperoxide.     

The electron spin relaxation of the electron acceptors of photosystem I reaction centre studied by microwave power saturation.

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electrom transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g=2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g=2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g=2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g=2.07 and 1.86 disappeared and new signals at g=1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B. These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the (4Fe-4S) CENTRes of the electron acceptors A and B resulted in saturation properties which are simular to those of the 2(4Fe-4S) ferredoxin from Clostridium pasteurianum. For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b=1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

The electron spin relaxation of the electron acceptors of Photosystem I reaction centre studied by microwave power saturation

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electron transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g = 2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g = 2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g = 2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g = 2.07 and 1.86 disappeared and new signals at g = 1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B.

These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the [4Fe-4S] centres of the electron acceptors A and B resulted in saturation properties which are similar to those of the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b = 1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.