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1.
The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process. 相似文献
2.
Cytochrome P-450scc-adrenodoxin interactions. Ionic effects on binding, and regulation of cytochrome reduction by bound steroid substrates 总被引:2,自引:0,他引:2
The binding of adrenodoxin to cytochrome P-450scc and the intracomplex electron transfer from the iron-sulfur center to the heme have been studied. Salt sensitivity of the protein complex suggests the participation of electrostatic forces, as is also seen for the complex of adrenodoxin with NADPH-adrenodoxin reductase. Differences in ion specificities for the complexes of adrenodoxin with the other two proteins suggest some differences in binding requirements. Insensitivity of the heme reduction to solution conditions (salt, detergent) and kinetic analysis indicate that the protein complex is formed rapidly and that intracomplex electron transfer then occurs more slowly. Factors governing the rate of this electron transfer were investigated; binding of a series of cholesterol derivatives was used to perturb the spin state, midpoint potential, and reduction rate of the heme, and thus to test for relationships among these parameters. A linear free energy relationship between the substrate-induced midpoint potential and reduction rate is seen, but none of the other parameters (including the strength of substrate binding) are correlated. Data indicate that factors other than spin state (i.e. steric requirements and bonding groups within the steroid-binding site) regulate the strength of steroid binding. The bound steroid then modulates both midpoint potential/reduction rate and spin state but by independent mechanisms. 相似文献
3.
Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Rainer Kümmerle Jacques Gaillard Panayotis Kyritsis Jean-Marc Moulis 《Journal of biological inorganic chemistry》2001,6(4):446-451
The semi-classical electron transfer theory has been very successful in describing reactions occurring in biological systems, but the relevant parameters in the case of iron-sulfur proteins remain unknown. The recent discovery that 2[4Fe-4S] proteins homologous to Chromatium vinosum ferredoxin contain clusters with different reduction potentials now gives the opportunity to study the dependence of the intramolecular electron transfer rate between these clusters as a function of the driving force. This work shows how decreasing the reduction potential difference between the clusters by site-directed mutagenesis of C. vinosum ferredoxin modifies the rate of electron hopping between the two redox sites of the protein by measuring the line broadening of selected 1H NMR signals. Beside the shifts of the reduction potentials, no signs of large structural changes or of significant alterations of the intrinsic kinetic parameters among the different variants of C. vinosum ferredoxin have been found. A reorganization energy of less than 0.5 eV was deduced from the dependence of the electron transfer rates with the reduction potential difference. This small value is associated with a weak electronic coupling between the two closely spaced clusters. This set of parameters, determined for the first time in an iron-sulfur protein, may help to explain how efficient vectorial electron transfer occurs with a small driving force in the many enzymatic systems containing a 2[4Fe-4S] domain. 相似文献
5.
Azurin contains two potential redox sites, a copper centre and, at the opposite end of the molecule, a cystine disulfide (RSSR). Intramolecular electron transfer between a pulse radiolytically produced RSSR- radical anion and the blue Cu(II) ion was studied in a series of azurins in which single-site mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half that of the corresponding wild-type azurin. In the His46Gly and His117Gly mutants, a water molecule is co-ordinated to the copper ion when no external ligands are added. Both these mutants also exhibit slower intramolecular electron transfer than the corresponding wild-type azurin. However, for the His117Gly mutant in the presence of excess imidazole, an azurin-imidazole complex is formed and the intramolecular electron-transfer rate increases considerably, becoming threefold faster than that observed in the native protein. Activation parameters for all these electron-transfer processes were determined and combined with data from earlier studies on intramolecular electron transfer in wild-type and single-site-mutated azurins. A linear relationship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force and possible electron-transfer pathways. 相似文献
6.
M C Walker J J Pueyo C Gómez-Moreno G Tollin 《Archives of biochemistry and biophysics》1990,281(1):76-83
The kinetics of reduction and intracomplex electron transfer in electrostatically stabilized and covalently crosslinked complexes between ferredoxin-NADP+ reductase (FNR) and flavodoxin (Fld) from the cyanobacterium Anabaena PCC 7119 were compared using laser flash photolysis. The second-order rate constant for reduction by 5-deazariboflavin semiquinone (dRfH) of FNR within the electrostatically stabilized complex at 10 mM ionic strength (4.0 X 10(8) M-1 s-1) was identical to that for free FNR. This suggests that the FAD cofactor of FNR is not sterically hindered upon complex formation. A lower limit of approximately 7000 s-1 was estimated for the first-order rate constant for intracomplex electron transfer from FNRred to Fldox under these conditions. In contrast, for the covalently crosslinked complex, a smaller second-order rate constant (2.1 X 10(8) M-1 s-1) was obtained for the reduction of FNR by dRfH within the complex, suggesting that some steric hindrance of the FAD cofactor of FNR occurs due to crosslinking. A limiting rate constant of 1000 s-1 for the intracomplex electron transfer reaction was obtained for the covalent complex, which was unaffected by changes in ionic strength. The substantially diminished limiting rate constant, relative to that of the electrostatic complex, may reflect either a suboptimal orientation of the redox cofactors within the covalent complex or a required structural reorganization preceding electron transfer which is not allowed once the proteins have been covalently linked. Thus, although the covalent complex is biochemically competent, it is not a quantitatively precise model for the catalytically relevant intermediate along the reaction pathway. 相似文献
7.
The details of most electron transfer reactions involving iron-sulfur proteins have remained undisclosed because of the lack of experimental methods suitable to measure precisely the relevant rates. Nuclear magnetic resonance (NMR) provides a powerful means to overcome these problems, at least with selected proteins. A combination of NMR studies and site-directed mutagenesis experiments has been instrumental in defining both the site of interaction and the main trends of the intracomplex electron transfer in the case of rubredoxin electron self-exchange. Analysis of the NMR data obtained for mixtures of different redox levels of several 2[4Fe-4S] ferredoxins provided both first-order, for intramolecular, and second-order, for intermolecular, rate constants. Their dependence as a function of structural changes gave insight into the mechanism of electron transfer in this type of protein. Contrary to some expectations, the high-spin [4Fe-4Se]+ clusters assembled in isopotential ferredoxins do not change the intramolecular electron transfer rate as compared to low-spin [4Fe-4S]+ homologs. In combination with activity measurements, the kinetic data have been used to model the electron transfer competent complexes between Clostridium pasteurianum ferredoxin and the main enzymes acting as redox partners in vivo. 相似文献
8.
M C Walker J J Pueyo J A Navarro C Gómez-Moreno G Tollin 《Archives of biochemistry and biophysics》1991,287(2):351-358
The influence of electrostatic forces on the formation of, and electron transfer within, transient complexes between redox proteins was examined by comparing ionic strength effects on the kinetics of the electron transfer reaction between reduced ferredoxins (Fd) and oxidized ferredoxin-NADP+ reductases (FNR) from Anabaena and from spinach, using laser flash photolysis techniques. With the Anabaena proteins, direct reduction by laser-generated flavin semiquinone of the FNR component was inhibited by complex formation at low ionic strength, whereas Fd reduction was not. The opposite results were obtained with the spinach system. These observations clearly indicate structural differences between the cyanobacterial and higher plant complexes. For the complex formed by the Anabaena proteins, the results indicate that electrostatic forces are not a major contributor to complex stability. However, the rate constant for intracomplex electron transfer had a biphasic dependence on ionic strength, suggesting that structural rearrangements within the transient complex facilitate electron transfer. In contrast to the Anabaena complex, electrostatic forces are important for the stabilization of the spinach Fd:FNR complex, and changes in ionic strength had little effect on the limiting rate constant for intracomplex electron transfer. This suggests that in this case the geometry of the initial collisional complex is optimal for reaction. These results provide a clear illustration of the differing roles that electrostatic interactions may play in controlling electron transfer between two redox proteins. 相似文献
9.
Visschers Ronald W. Vulto Simone I.E. Jones Michael R. Grondelle Rienk van Kraayenhof Ruud 《Photosynthesis research》1999,59(1):95-104
The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane. 相似文献
10.
Cytochrome c-552 from Chromatium vinosum is an unusual heme protein in that it contains two hemes and one flavin per molecule. To investigate whether intramolecular electron transfer occurs in this protein, we have studied its reduction by external photoreduced flavin by using pulsed-laser excitation. This approach allows us to measure reduction kinetics on the mirosecond time scale. Both fully reduced lumiflavin and lumiflavin semiquinone radical reduce cytochrome c-552 with second-order rate constants of approximately 1.4 x 10(6) M-1s-1 and 1.9 x 10(8) M-1 s-1, respectively. Kinetic and spectral data and the results of similar studies with riboflavin indicate that both the flavin and heme moieties of cytochrome c-552 are reduced simultaneously on a millisecond time scale, with the transient formation of a protein-bound flavin anion radical. This is suggested to be due to rapid intramolecular electron transfer. Further, steric restrictions play an important role in the reduction reaction. Studies were conducted on the redox processes following photolysis of CO-ferrocytochrome c-552 in which the flavin was partly oxidized to resolve the kinetics of electron transfer between the heme and flavin of cytochrome c-552. Based on these results, we conclude that intramolecular electron transfer from ferrous heme to oxidized flavin occurs with a first-order rate constant of greater than 1.4 x 10(6) s-1. 相似文献
11.
Transient kinetics of reduction of zucchini squash ascorbate oxidase (AO) by lumiflavin semiquinone have been studied by using laser flash photolysis. Second-order kinetics were obtained for reduction of the type I copper with a rate constant of 2.7 X 10(7) M-1 s-1, which is comparable to that obtained with other blue copper proteins such as plastocyanin. Following reduction, the type I copper was reoxidized in a protein concentration independent (i.e., intramolecular) reaction (kobs = 160 s-1). Comparison with literature values for limiting rate constants in transient single-turnover kinetic experiments suggests that intramolecular electron transfer probably is the rate-limiting step in enzyme catalysis. The extent of reoxidation of type I copper was approximately 55%, which is consistent with the approximately equal redox potentials of the type I and type III copper centers. Neither azide nor fluoride caused any significant changes in kinetics, although they are enzyme inhibitors and are thought to bind to the type II copper. In contrast, cyanide caused a concentration-dependent decrease in the extent of intramolecular electron transfer (with no change in rate constant), and decreased the rate constant for reduction of the type I copper by a factor of 2. The apparent dissociation constant for cyanide (0.2-0.4 mM) is similar to that reported for inhibition of enzyme activity. Removal of the type II copper from AO only marginally affected the kinetics of electron transfer to type I copper (k = 3.2 x 10(7) M-1 s-1) and slightly increased the extent but did not alter the rate constant of intramolecular electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Effects of Temperature and ΔG° on Electron Transfer from Cytochrome c2 to the Photosynthetic Reaction Center of the Purple Bacterium Rhodobacter sphaeroides
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G. Venturoli F. Drepper J.C. Williams J.P. Allen X. Lin P. Mathis 《Biophysical journal》1998,74(6):3226-3240
The kinetics of electron transfer from cytochrome c2 to the primary donor (P) of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated by time-resolved absorption spectroscopy. Rereduction of P+ induced by a laser pulse has been measured at temperatures from 300 K to 220 K in a series of specifically mutated reaction centers characterized by altered midpoint redox potentials of P+/P varying from 410 mV to 765 mV (as compared to 505 mV for wild type). Rate constants for first-order electron donation within preformed reaction center–cytochrome c2 complexes and for the bimolecular oxidation of free cytochrome c2 have been obtained by multiexponential deconvolution of the kinetics. At all temperatures the rate of the fastest intracomplex electron transfer increases by more than two orders of magnitude as the driving force −ΔG° is varied over a range of 350 meV. The temperature and ΔG° dependences of the rate constant fit the Marcus equation well. Global analysis yields a reorganization energy λ = 0.96 ± 0.07 eV and a set of electronic matrix elements, specific for each mutant, ranging from 1.2 10−4 eV to 2.5 10−4 eV. Analysis in terms of the Jortner equation indicates that the best fit is obtained in the classical limit and restricts the range of coupled vibrational modes to frequencies lower than ∼200 cm−1. An additional slower kinetic component of P+ reduction, attributed to electron transfer from cyt c2 docked in a nonoptimal configuration of the complex, displays a Marcus type dependence of the rate constant upon ΔG°, characterized by a similar value of λ (0.8 ± 0.1 eV) and by an average electronic matrix element smaller by more than one order of magnitude. In all of the mutants, as the temperature is decreased below 260 K, both intracomplex reactions are abruptly inhibited, their rate being negligible at 220 K. The free energy dependence of the second-order rate constant for oxidation of cyt c2 in solution suggests that the collisional reaction is partially diffusion controlled, reaching the diffusion limit at exothermicities between 150 and 250 meV over the temperature range investigated. 相似文献
13.
A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction. 相似文献
14.
C T Przysiecki A K Bhattacharyya G Tollin M A Cusanovich 《The Journal of biological chemistry》1985,260(3):1452-1458
The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1). 相似文献
15.
Bill Durham Jill L. Fairris Mark McLean Frank Millett Jill R. Scott Stephen G. Sligar Anne Willie 《Journal of bioenergetics and biomembranes》1995,27(3):331-340
The reaction of cytochromeb
5 with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments. 相似文献
16.
The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first
in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally
well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants
and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated
by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large
number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally
well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the
results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism.
Received: 2 January 1997 / Accepted: 6 February 1997 相似文献
17.
Matthew C. F. Wander James D. Kubicki Martin A. A. Schoonen 《Origins of life and evolution of the biosphere》2008,38(3):195-209
Nitrogen reduction by ferrous iron has been suggested as an important mechanism in the formation of ammonia on pre-biotic Earth. This paper examines the effects of adsorption of ferrous iron onto a goethite (alpha-FeOOH) substrate on the thermodynamic driving force and rate of a ferrous iron-mediated reduction of N2 as compared with the homogeneous aqueous reaction. Utilizing density functional theory and Marcus Theory of proton coupled electron transfer reactions, the following two reactions were studied: Fe2+aq + N2aq + H2Oaq --> N2H* + FeOH2+aq and triple bond Fe2+ads + N2aq + 2H2Oaq --> N2H* + alpha-FeOOHs + 2H+aq. Although the rates of both reactions were calculated to be approximately zero at 298 K, the model results suggest that adsorption alters the thermodynamic driving force for the reaction but has no other effect on the direct electron transfer kinetics. Given that simply altering the thermodynamic driving force will not reduce dinitrogen, we can make mechanistic connections between possible prebiotic pathways and biological N2 reduction. The key to reduction in both cases is N2 adsorption to multiple transition metal centers with competitive H2 production. 相似文献
18.
The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved. 相似文献
19.
Balashov SP 《Biochimica et biophysica acta》2000,1460(1):75-94
Light-induced changes of the proton affinities of amino acid side groups are the driving force for proton translocation in bacteriorhodopsin. Recent progress in obtaining structures of bacteriorhodopsin and its intermediates with an increasingly higher resolution, together with functional studies utilizing mutant pigments and spectroscopic methods, have provided important information on the molecular architecture of the proton transfer pathways and the key groups involved in proton transport. In the present paper I consider mechanisms of light-induced proton release and uptake and intramolecular proton transport and mechanisms of modulation of proton affinities of key groups in the framework of these data. Special attention is given to some important aspects that have surfaced recently. These are the coupling of protonation states of groups involved in proton transport, the complex titration of the counterion to the Schiff base and its origin, the role of the transient protonation of buried groups in catalysis of the chromophore's thermal isomerization, and the relationship between proton affinities of the groups and the pH dependencies of the rate constants of the photocycle and proton transfer reactions. 相似文献
20.
Min T Ergenekan CE Eidsness MK Ichiye T Kang C 《Protein science : a publication of the Protein Society》2001,10(3):613-621
Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe-S] cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin. 相似文献