Dynamic docking and electron-transfer between cytochrome b5 and a suite of myoglobin surface-charge mutants. Introduction of a functional-docking algorithm for protein-protein complexes

Horse myoglobin (Mb) provides a convenient "workbench" for probing the effects of electrostatics on binding and reactivity in the dynamic [Mb, cytochrome b(5)] electron-transfer (ET) complex. We have combined mutagenesis and heme neutralization to prepare a suite of six Mb surface-charge variants: the [S92D]Mb and [V67R]Mb mutants introduce additional charges on the "front" face, and incorporation of the heme di-ester into each of these neutralizes the charge on the heme propionates which further increases the positive charge on the "front" face. For this set of mutants, the nominal charge of Mb changes by -1 to +3 units relative to that for native Mb. For each member of this set, we have measured the bimolecular quenching rate constant (k(2)) for the photoinitiated (3)ZnDMb --> Fe(3+)b(5) ET reaction as a function of ionic strength. We find: (i) a dramatic decoupling of binding and reactivity, in which k(2) varies approximately 10(3)-fold within the suite of Mbs without a significant change in binding affinity; (ii) the ET reaction occurs within the "thermodynamic" or "rapid exchange" limit of the "Dynamic Docking" model, in which a large ensemble of weakly bound protein-protein configurations contribute to binding, but only a few are reactive, as shown by the fact that the zero-ionic-strength bimolecular rate constant varies exponentially with the net charge on Mb; (iii) Brownian dynamic docking profiles allow us to visualize the microscopic basis of dynamic docking. To describe these results we present a new theoretical approach which mathematically combines PATHWAY donor/acceptor coupling calculations with Poisson-Boltzmann-based electrostatics estimates of the docking energetics in a Monte Carlo (MC) sampling framework that is thus specially tailored to the intermolecular ET problem. This procedure is extremely efficient because it targets only the functionally active complex geometries by introducing a "reactivity filter" into the computations themselves, rather than as a subsequent step. This efficiency allows us to employ more computationally expensive and accurate methods to describe the relevant intermolecular interaction energies and the protein-mediated donor/acceptor coupling interactions. It is employed here to compute the changes in the bimolecular rate constant for ET between Mb and cyt b(5) upon variations in the myoglobin surface charge, pH, and ionic strength.     

Hopping in the electron-transfer photocycle of the 1:1 complex of Zn-cytochrome c peroxidase with cytochrome c

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin pi-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein. I, in general, is thought to return to the ground state by a thermal ET process that involves direct heme-heme back-ET to complete a simple photocycle. We here contrast intracomplex ET between yeast iso-1 Cc and ZnCcP(WT) (wild-type) with that for two ZnCcP(X) variants: X = W191F, with redox-active W191 replaced by Phe; WYM4, a W191F mutant with further replacement of four other potentially redox-active sites (W51F, Y187F, Y229F, and Y236F). The results show that W191 acts as an ET mediator, which "short-circuits" the direct heme-heme back-ET through a two-step, hopping process in which the ZnP+ cation radical formed by photoinitiated ET rapidly oxidizes W191, and the resultant W191+, in turn, rapidly oxidizes Fe2+Cc.     

Measurement of the forward and back rate constants for electron transfer at the interface between two immiscible electrolyte solutions using scanning electrochemical microscopy (SECM): Theory and experiment

A new numerical model is developed for the scanning electrochemical microscopy (SECM) feedback mode for reversible electron transfer (ET) processes at the interface between two immiscible electrolyte solutions (ITIES). Results from this model were compared with data obtained using an earlier SECM feedback model in which the back reaction was not considered, to identify when the latter will be important. The dependence of the ET rate constant for the oxidation of 7,7,8,8-tetracyanoquinodimethane radical anion (TCNQ⁻) in 1,2-dichloroethane (DCE) by aqueous ferricyanide on the interfacial potential drop (Δ_w^oφ) was studied using SECM. The Δ_w^oφ value was varied by changing the concentration of NaClO₄ in the aqueous phase while a fixed concentration of organic electrolyte, tetra-n-hexylammonium perchlorate, was used in the DCE phase. The results obtained were compared to earlier published studies on the forward reaction between TCNQ in DCE and aqueous ferrocyanide. Both the forward and back ET rate constants were found to depend strongly on the interfacial potential drop, with measured ET coefficients in the region of 0.5–0.6. A similar ET rate constant was observed at zero driving force for both the forward and back reactions. These experimental results suggest that the Butler–Volmer model applies to ET at the ITIES, when the driving force for the reaction is low, and under conditions of relatively high ionic strength in both the aqueous and organic phases.     

Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana

The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD_ox), semiquinone form (FADH), and anionic hydroquinone form (FAD_redH⁻). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD_ox to the anionic semiquinone form (FAD⁻) with a quantum efficiency of about 2% and a back recovery time of about 10 s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD_ox to FADH, FADH to FAD_redH⁻, and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.     

Comparative study of electron transfer reactions at the ionic liquid/water and organic/water interfaces

The rates of electron transfer (ET) reactions at the water/ionic liquid (IL) interface have been measured for the first time using scanning electrochemical microscopy. The standard bimolecular rate constant of the interfacial ET between ferrocene dissolved in 1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide and aqueous ferricyanide (0.4 M-1 cm s-1) was found to be approximately 30 times higher than the corresponding rate constant measured at the water/1,2-dichloroethane interface. The driving force dependence of the ET rate was investigated over a wide range of the interfacial potential drop values (>200 mV). The observed Butler-Volmer-type dependence is discussed in terms of the interfacial model. The ET was also probed at the interface between aqueous solution and the mixture of the IL and 1,2-dichloroethane. The mole fractions in this mixture were varied systematically to investigate the transition from the water/organic to the water/IL interface. The observed decrease in the rate constant with increasing mole fraction of 1,2-dichloroethane is in contrast with the previously reported direct correlation between the electrochemical rate constant and the diffusion coefficient of redox species in solution.     

Following evolution of bacteriorhodopsin in its reactive excited state via stimulated emission pumping

New information concerning the photochemical dynamics of bacteriorhodopsin (BR) is obtained by impulsively stimulating emission from the reactive fluorescent state. Depletion of the excited-state fluorescence leads to an equal reduction in production of later photoproducts. Accordingly, chromophores which are forced back to the ground state via emission do not continue on in the photocycle, conclusively demonstrating that the fluorescent state is a photocycle intermediate. The insensitivity of depletion dynamics to the "dump" pulse timing, throughout the fluorescent states lifetime, and the biological inactivity of the dumped population suggest that the fluorescent-state structure is constant, well-defined, and significantly different than that where crossing to the ground state takes place naturally. In conjunction with conclusions from comparing the photophysics of BR with those of synthetic analogues containing "locked" retinals, present results show that large-amplitude torsion around C13=C14 is required to go between the above structures.     

Photoinduced intramolecular electron-transfer reactions of reconstituted met- and zinc-myoglobins appending acridine and methylacridinium ion as DNA-binders

Three types of reconstituted met- and zinc-myoglobin (metMb and ZnMb) dyads, ZnMbAc(4)Me+, ZnMbAc(6)Me+, and metMbAc(6) have been prepared by incorporating chemically modified metalloporphyrin cofactor appending an acridine (Ac) or a methylacridinium ion ([AcMe]+) into apo-Mb. In the bimolecular system between ZnMb and [AcMe]+, the photoexcited triplet state of ZnMb, 3(ZnMb)*, was successfully quenched by [AcMe]+ to form the radical pair of ZnMb cation (ZnMb*+) and reduced methylacridine ([AcMe]*), followed by a thermal back ET reaction. The rate constants for the intermolecular quenching ET (kq) and the back ET reaction (kb) at 25 degrees C were successfully obtained as kq = (8.8 +/- 0.4) x 10(7) M(-1) s(-1) and kb = (1.2 +/- 0.1) x 10(8) M(-1) s(-1), respectively. On the other hand, in case of the intramolecular photoinduced ET reactions of ZnMbAc(4)Me+ and ZnMbAc(6)Me+ dyads, the first-order quenching rate constants (kET) of 3(ZnMb)* by [AcMe]+ moiety were determined to be kET = 2.6 x 10(3) and 2.5 x 10(3) s(-1), respectively. When such ET occurs along the alkyl spacer via through-bond mechanism at the surface of Mb, the obtained kET is reasonable to provide decay constant of beta (1.0-1.3 A(-1)). Upon photoirradiation of [AcMe]+ moiety, kinetic studies also presented the intramolecular quenching reactions from the excited singlet state, 1([AcMe]+)*, whose likely process is the photoinduced energy-transfer reaction. For metMbAc(6) dyad, steady-state fluorescence was almost quenched, while the signal around 440 nm gradually appeared in the presence of various concentrations of DNA. Our study implies that synthetic manipulation at the Mb surface, by using an artificial DNA-binder coupled with photoinduced reaction, may provide valuable information to construct new Mb-DNA complex and sensitive fluorescent for DNA.     

Photoinduced electron transfer and geminate recombination in liquids on short time scales: Experiments and theory

The coupled processes of intermolecular photoinduced forward electron transfer and geminate recombination between the (hole) donor (Rhodamine 3B) and (hole) acceptors (N,N-dimethylaniline) are studied in three molecular liquids: acetonitrile, butyronitrile, and benzonitrile. Two color pump-probe experiments on time scales from approximately 100 fs to hundreds of picoseconds give information about the depletion of the donor excited state due to forward electron transfer and the survival kinetics of the radicals produced by forward electron transfer. The data are analyzed with a model presented previously that includes distance dependent forward and back electron transfer rates, donor and acceptor diffusion, solvent structure, and the hydrodynamic effect in a mean-field theory of through solvent electron transfer. The forward electron transfer is in the normal regime, and the Marcus equation for the distance dependence of the transfer rate is used. The forward electron transfer data for several concentrations in the three solvents are fitted to the theory with a single adjustable parameter, the electronic coupling matrix element Jf at contact. Within experimental error all concentrations in all three solvents are fitted with the same value of Jf. The geminate recombination (back transfer) is in the inverted region, and semiclassical treatment developed by Jortner [J. Chem. Phys. 64, 4860 (1976)] is used to describe the distance dependence of the back electron transfer. The data are fitted with the single adjustable parameter Jb. It is found that the value of Jb decreases as the solvent viscosity increases. Possible explanations are discussed.     

Metal ion-dependent fluorescent dynamics of photoexcited zinc-porphyrin and zinc-myoglobin modified with ethylenediaminetetraacetic acid

The reconstituted zinc-myoglobin (ZnMb) dyads, ZnMb-[M(II)(edta)], have been prepared by incorporating a zinc-porphyrin (ZnP) cofactor modified with ethylenediaminetetraacetic acid (H(4)edta) into apo-Mb. In case of the monomeric ZnP(edta) cofactor coordinated by one pyridine molecule, ZnP(py)(edta), a spontaneous 1:1 complex with a transient metal ion was formed in an aqueous solvent, and the photoexcited singlet state of ZnP, (1)(ZnP)*, was quenched by the [Cu(II)(edta)] moiety through intramolecular photoinduced electron-transfer (ET) reaction. The rate constant for the intramolecular quenching ET (k(q)) at 25 degrees C was successfully obtained as k(q) = 5.1 x 10(9) s(-1). In the case of Co(2+), Ni(2+), and Mn(2+), intersystem crossing by paramagnetic effect was mainly considered between (1)(ZnP)* and the [M(II)(edta)] complex. For the ZnMb-[M(II)(edta)] systems, the intramolecular ET reaction between the excited singlet state of (1)(ZnMb)* and the [Cu(II)(edta)] moieties provided the slower quenching rate constant, k(q) = 2.1 x 10(8) s(-1), compared with that of the ZnP(py)(edta) one. Kinetic studies also presented the efficient fluorescence quenching of the (1)(ZnMb)*-[Co(II)(edta)] dyad. Our study clearly demonstrates that wrapping of the ZnP cofactor by the apoprotein matrix and synthetic manipulation at the Mb surface ensure metal ion-sensitive fluorescent dynamics of ZnMb and provides valuable information to elucidate the complicated mechanism of the biological photoinduced ET reactions of hemoproteins.     

Revealing the influence of the strength of coulomb interactions on the viscosity and interfacial tension of ionic liquid cosolvent mixtures

Within this work, viscosity and interfacial tension of selected ionic liquid cosolvent mixtures, [EMIM][EtOSO3] (1-ethyl-3-methyl-immidazolium ethyl sulfate) with water and ethanol, were studied as a function of composition by surface light scattering (SLS) and the pendant drop method in a consistent manner, allowing a close insight into the nature of interactions. Here, we show that the viscosity behavior clearly reflects the bulk structure of the ionic liquid cosolvent mixtures and correlates to the fluid structure at the phase boundary. In contrast to former work, we found the viscosity of ionic liquid [EMIM][EtOSO3] to be decreasing the stronger by small amounts of the cosolvents and the lower their dielectric constant. Furthermore, two distinct trends for the dependence of the viscosity on the cosolvent concentration were resolved. These were assigned to ion-dipole interactions dominating in the salt-rich region and to dipole-dipole interactions in the diluted one. A crossover between both regions is reflected by the interfacial tension data, where it seems that up to a "critical" concentration almost no cosolvent is present at the phase boundary.     

The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin: mechanism and evidence for two M species

The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin is investigated both for the native pigment and its D96N mutant. The experimental setup is based on creating the M intermediate by a first pulse, followed by a (blue) laser pulse which drives the back photoreaction of M. Experiments are carried out varying the delay between the two pulses, as well as the temperature over the -25 degrees C-20 degrees C range. It is found that the kinetic patterns of the M back photoreaction change with time after the generation of this intermediate. The data provide independent evidence for the suggestion of a photocycle mechanism based on two distinct M intermediates. They are thus in keeping with the consecutive model of Varo and Lanyi (Biochemistry 30, 5016-5022; 1991), although they cannot exclude other models such as those based on branched or parallel cycles. More generally, we offer a "photochemical" approach to discriminating between intermediate stages in the photocycle which does not depend on spectroscopic and/or kinetic data. While markedly affecting the rate of the M --> N transition in the photocycle, the rate of the thermal step in back photoreaction of M, at both room and low temperatures, is not significantly affected by the D96N mutation. It is proposed that while Asp 96 is the Schiff-base protonating moiety in the M --> N transition, another residue (most probably Asp 85) reprotonates the Schiff base following light absorption by M.     

A true electron-transfer reaction between 5,10,15,20-tetraphenylporphyrinato cadmium(II) and the hexacyanoferrate couple at the nitrobenzene/water interface.

The ability of some metal complexes of 5,10,15,20-tetraphenylporphyrin (TPP) to give a voltammetric wave due to the heterogeneous electron transfer (ET) at a nitrobenzene (NB)/water (W) interface has been examined. The previously-proposed, electron-conductor separating oil-water (ECSOW) system has been successfully employed to find that the TPP complex with cadmium(II) added to NB gives a well-defined, reversible wave for the heterogeneous (i.e., "true") ET with the hexacyanoferrate couple in W. A digital simulation analysis has entirely excluded the possibility of the ion-transfer mechanism due to the homogeneous ET in W. The a.c. impedance method has then been used to determine the kinetic parameters including the standard rate constant k0 (= 0.10 cm M(-1) s(-1)) and the transfer coefficient alpha (= 0.53 at the half-wave potential). These values are in good agreement with those predicted from the Marcus theory with the assumption that the heterogeneous ET due to molecular collision occurs at the "sharp" NB/W interface.     

trans-[(en)~2(NO~2)Co(O~2CC~5H~5N)]^2^+/Fe(Ⅱ)间电子转移反应动力学及机理研究

合成了新型Co(Ⅲ)配合物trans-[(en)~2(NO~2)Co(O~2CC~5H~5N)](ClO~4)~2, 并通过紫外可见光谱、红外光谱、元素分析和X射线单晶衍射分析进行了表征。同时分别以[Fe(CN)~6]^4^-和[Fe(CN)~5(H~2O)]^3^-作为还原剂, 考察了该配合物被还原的反应动力学行为。结果表明两反应体系分别按外配位界机理和内配位界机理进行电子传递。在25℃, Ⅰ=0.5mol·L^-^1,trans-[(en)~2(NO~2)Co(O~2CC~5H~5N)]^2^+/[Fe(CN)~6]^4^-反应体系的前驱配合物离子对形成常数Q~i~p=29mol^-^1·L, 电子转移速率常数k~e~t=2.4×10^-^4s^-^1,电子转移过程的活化焓△H^≠~e~t和活化熵△S^≠~e~t分别为1.2×10^2kJ·mol^-^1和5.0×10^2J·mol^-^1·K^-^1。在40℃, pH=8.0, Ⅰ=0.1mol·L^-^1,trans-[(en)~2(NO~2)Co(O~2CC~5H~4N)]^2^+/[Fe(CN)~5(H~2O)]^3^-反应体系前驱双核配合物分子内电子转移速率常数为7.0×10^-^5s^-^1。最后讨论了分子轨道对称性, 两金属中心氧化还原电势差等因素对电子转移速率的影响。     

Intramolecular electron and energy transfer in an axial ZnP-pyridylfullerene complex as studied by X- and W-band time-resolved EPR spectroscopy

Light-driven electron transfer (ET) and energy transfer (EnT) in a self-assembled via axial coordination Zn-porphyrin-pyridylfullerene (ZnP-PyrF) complex were studied by time-resolved electron paramagnetic resonance (TREPR) spectroscopy at 9.5 GHz (X-band) and 95 GHz (W-band). The studies over a wide temperature range were carried out in media of different polarity, including isotropic toluene and tetrahydrofuran (THF), and anisotropic nematic liquid crystals (LCs), E-7 and ZLI-4389. At low temperatures (frozen matrices), photoexcitation of the ZnP donor results mainly in singlet-singlet EnT to the pyridine-appended fullerene acceptor. In fluid phases ET is the dominant process. Specifically, in isotropic solvents the generated radical pairs (RPs) are long-lived, with lifetimes exceeding that observed for covalently linked donor-acceptor systems. It is concluded that in liquid phases of both polar and nonpolar solvents the separation of the tightly bound complex into the more loosely bound structure slows down the back ET (BET) process. Photoexcitation of the donor in fluid phases of LCs does not result in the creation of the long-lived RPs, since the ordered LC matrix hinders the separation of the complex constituents. As a result, fast intramolecular BET takes place in the tightly bound complex. Contrarily to the behavior of covalently linked donor-acceptor systems in different LCs, the polarity of the LC matrix affects the ET process. Moreover, in contrast to covalently linked D-s-A systems, utilization of LCs for the coordinatively linked D-s-A complexes does not reduce the ET rates significantly.     

Electron-transfer chemistry of Ru-linker-(heme)-modified myoglobin: rapid intraprotein reduction of a photogenerated porphyrin cation radical

We report the synthesis and characterization of RuC7, a complex in which a heme is covalently attached to a [Ru(bpy)(3)](2+) complex through a -(CH(2))(7)- linker. Insertion of RuC7 into horse heart apomyoglobin gives RuC7Mb, a Ru(heme)-protein conjugate in which [Ru(bpy)(3)](2+) emission is highly quenched. The rate of photoinduced electron transfer (ET) from the resting (Ru(2+)/Fe(3+)) to the transient (Ru(3+)/Fe(2+)) state of RuC7Mb is >10(8) s(-1); the back ET rate (to regenerate Ru(2+)/Fe(3+)) is 1.4 x 10(7) s(-1). Irreversible oxidative quenching by [Co(NH(3))(5)Cl](2+) generates Ru(3+)/Fe(3+): the Ru(3+) complex then oxidizes the porphyrin to a cation radical (P*+); in a subsequent step, P*+ oxidizes both Fe(3+) (to give Fe(IV)=O) and an amino acid residue. The rate of intramolecular reduction of P*+ is 9.8 x 10(3) s(-1); the rate of ferryl formation is 2.9 x 10(3) s(-1). Strong EPR signals attributable to tyrosine and tryptophan radicals were recorded after RuC7MbM(3+) (M = Fe, Mn) was flash-quenched/frozen.     

Accelerating and decelerating effects of metal ions on electron-transfer reduction of quinones as a function of temperature and binding modes of metal ions to semiquinone radical anions

The accelerating effect of Sc(3+) on the electron-transfer (ET) reduction of the p-benzoquinone derivative 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ) by 10,10'-dimethyl-9,9'-biacridine ((AcrH)(2)) at 233 K changes to a decelerating effect with increasing reaction temperature; the observed second-order rate constant k(et) decreases with increasing Sc(3+) concentration at high concentrations of Sc(3+) at 298 K. At 263 K the k(et) value remains constant with increasing Sc(3+) concentration. Such a remarkable difference with regard to dependence of k(et) on [Sc(3+)] between low and high temperatures results from the difference in relative activity of two ET pathways that depend on temperature, one of which affords 1:1 complex TolSQ*(-)-Sc(3+), and the other 1:2 complex TolSQ*(-)-(Sc(3+))(2) with additional binding of Sc(3+) to TolSQ*(-)-Sc(3+). The formation of TolSQ*(-)-Sc(3+) and TolSQ*(-)-(Sc(3+))(2) complexes was confirmed by EPR spectroscopy in the ET reduction of TolSQ in the presence of low and high concentrations of Sc(3+), respectively. The effects of metal ions on other ET reactions of quinones to afford 1:1 and 1:2 complexes between semiquinone radical anions and metal ions are also reported. The ET pathway affording the 1:2 complexes has smaller activation enthalpies DeltaH( not equal) and more negative activation entropies DeltaS( not equal) because of stronger binding of metal ions and more restricted geometries of the ET transition states as compared with the ET pathway to afford the 1:1 complexes.     

Dynamic docking of cytochrome b5 with myoglobin and alpha-hemoglobin: heme-neutralization "squares" and the binding of electron-transfer-reactive configurations

Intracomplex electron transfer (ET) occurs most often in intrinsically transient, low affinity complexes. As a result, the means by which adequate specificity and reactivity are obtained to support effective ET is still poorly understood. We report here on two such ET complexes: cytochrome b5 (cyt b5) in reaction with its physiological partners, myoglobin (Mb) and hemoglobin (Hb). These complexes obey the Dynamic Docking (DD) paradigm: a large ensemble of weakly bound protein-protein configurations contribute to binding in the rapid-exchange limit, but only a few are ET-active. We report the ionic-strength dependence of the second-order rate constant, k2, for photoinitiated ET from within all four combinations of heme-neutralized Zn deuteroporphyrin-substituted Mb/alphaHb undergoing ET with cyt b5, the four "corners" of a "heme-neutralization square". These experiments provide insights into the relative importance of both global and local electrostatic contributions to the binding of reactive configurations, which are too few to be observed directly. To interpret the variations of k2 arising from heme neutralization, we have developed a procedure by which comparisons of the ET rate constants for a heme-neutralization square permit us to decompose the free energy of reactive binding into individual local electrostatic contributions associated with interactions between (i) the propionates of the two hemes and (ii) the heme of each protein with the polypeptide of its partner. Most notably, we find the contribution from the repulsion between propionates of partner hemes to the reactive binding free energy to be surprisingly small, DeltaG(Hb) approximately +1 kcal/mol at ambient temperature, 18 mM ionic strength, and we speculate about possible causes of this observation. To confirm the fundamental assumption of these studies, that the structure of a heme-neutralized protein is unaltered either by substitution of Zn or by heme neutralization, we have obtained the X-ray structure of ZnMb prepared with the porphyrin dimethyl ester and find it to be nearly isostructural with the native protein.     

Catalytic effects of dioxygen on intramolecular electron transfer in radical ion pairs of zinc porphyrin-linked fullerenes.

Dioxygen accelerates back electron transfer (BET) processes between a fullerene radical anion (C60) and a radical cation of zinc porphyrin (ZnP) in photolytically generated ZnP.+-C60.- and ZnP.+-H2P-C60.- radical ion pairs. The rate constant of BET increases linearly with increasing oxygen concentration without, however, forming reactive oxygen species, such as singlet oxygen or superoxide anion. When ferrocene (Fc) is used as a terminal electron donor moiety instead of ZnP (i.e., Fc-ZnP-C60), no catalytic effects of dioxygen were, however, observed for the BET in Fc+-ZnP-C60.-, that is, from C60.- to the ferricenium ion. In the case of ZnP-containing C60 systems, the partial coordination of O2 to ZnP.+ facilitates an intermolecular electron transfer (ET) from C60.- to O2. This rate-determining ET step is followed by a rapid intramolecular ET from O2.- to ZnP.+ in the corresponding O2.--ZnP.+ complex and hereby regenerating O2. In summary, O2 acts as a novel catalyst in accelerating the BET of the C60.--ZnP.+ radical ion pairs.     

Isomerization of DNA-bound distilbazolium ligand induced by electron transfer from photoexcited tris(1,10-phenanthroline)Ru(II)

The ability of the DNA duplex to behave as an efficient organized medium for cis-trans isomerization induced by electron transfer (ET) has been explored. Isomerization studies, luminescence quenching and DNA photocleavage assays show that photoexcited Ru(1,10-phenanthroline [phen])3(2+) transfers an electron to E,Z1,4-bis[2-(1-methylpyridinium-4-yl)vinyl]benzene (E,Z pMPVB), which subsequently undergoes one-way isomerization to E,E pMPVB. The unusual feature of the system is manifested by the lack of friction that is usually imposed on the photoisomerizable ligand by highly organized media. The apparent rate of ET in DNA increases when compared with the homogeneous solution. However, after correction for the local concentration of the reagents onto the biopolymer, the rate constant becomes independent of the DNA concentration and is at least 4 x 10(2) times smaller than that in the homogeneous aqueous solution. Using the photoinduced isomerization system, a large enhancement in the efficiency of single-strand break formation was found in plasmid DNA over that for Ru(phen)3(+2) alone using irradiation at lambda > 480 nm.