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Cytochrome b2, an electron carrier between flavocytochrome b2 and cytochrome c. Rapid kinetic characterization of the electron-transfer parameters with ionic-strength-dependence.

ABSTRACT: The oxidation-reduction properties of free cytochrome b2 isolated by controlled proteolysis from flavocytochrome b2, i.e. the flavodehydrogenase-bound cytochrome b2, were investigated by using stopped-flow spectrophotometry. The rapid kinetics of the reduction of cytochrome b2 by flavocytochrome b2 in the presence of L-lactate are reported. The self-exchange rate constant between reduced cytochrome b2 bound to the flavodehydrogenase and free cytochrome b2 was determined to be 10(5) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. The specific electron-transfer reaction between reduced cytochrome b2 and cytochrome c was also studied, giving an apparent second-order rate constant of 10(7) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. This electron-exchange rate is slightly modulated by ionic strength, following the Debye-Hückel relationship with a charge factor Z1Z2 = -1.9. Comparison of these data with those for the reduction of cytochrome c by flavodehydrogenase-bound cytochrome b2 [Capeillère-Blandin (1982) Eur. J. Biochem. 128, 533-542] leads to the conclusion that the intramolecular electron exchange between haem b2 and haem c within the reaction complex occurs at a rate very similar to that determined experimentally in presence of the flavodehydrogenase domain. The low reaction rate observed with free cytochrome b2 is ascribed to the low stability of the reaction complex formed between free cytochrome b2 and cytochrome c.

SUBMITTER: Capeillere-Blandin C

PROVIDER: S-EPMC1148095 | biostudies-other | 1987 Jul

REPOSITORIES: biostudies-other

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Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2.

Project description:The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

| S-EPMC1132764 | biostudies-other

Modulation of flavocytochrome b2 intraprotein electron transfer via an interdomain hinge region.

Project description:The two domains of flavocytochrome b2 are connected by a typical hinge peptide. To probe the role of the hinge in modulating the efficiency of intraprotein electron transfer between these two domains, a number of mutant enzymes with truncated hinge regions were previously constructed and characterized [Sharp, Chapman and Reid (1996) Biochemistry 35, 891-899]. In the present study two mutant enzymes with elongated hinge regions have been constructed (HI3 and HI6) to further our understanding of the controlling influence of hinge length and primary structure on intraprotein electron transfer. Modification of the hinge had little effect on the lactate dehydrogenase activity of the enzyme, as was evident from steady-state experiments using ferricyanide as electron acceptor and from pre-steady-state experiments monitoring flavin reduction. However, the hinge insertions lowered the enzyme's effectiveness as a cytochrome c reductase. This effect results from a defect at the first interdomain electron-transfer step (FMNH2 --> haem electron transfer), where the rate constants for haem reduction in HI3 and HI6 were 50- and 100-fold lower than the corresponding value for the wild-type enzyme. Preservation of structural integrity within the hinge region is apparently essential for efficient intraprotein electron transfer.

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The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2.

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| S-EPMC1132485 | biostudies-other

Electron transfer from cytochrome c(2) to the reaction center: a transition state model for ionic strength effects due to neutral mutations.

Project description:Interprotein electron transfer plays an important role in biological energy conversion. In this work, the electron transfer reaction between cytochrome c(2) (cyt) and the reaction center (RC) was studied to determine the mechanisms coupling association and electron transfer. Previous studies have shown that mutation of hydrophobic residues in the reaction interface, particularly Tyr L162, changes the binding affinity and rates of electron transfer at low ionic strengths. In this study, the effect of ionic strength on the second-order electron transfer rate constant, k(2), between cyt c(2) and native or mutant RCs was examined. Mutations of hydrophobic and hydrogen bonding residues caused k(2) to decrease more rapidly with an increase in ionic strength. This change is explained with a transition state model by a switch from a diffusion-limited reaction in native RCs, where electron transfer occurs upon each binding event, to a fast exchange reaction in the Tyr L162 mutant, where dissociation occurs before electron transfer and k(2) depends upon the equilibrium between bound and free protein complexes. The difference in ionic strength dependence is attributed to a smaller effect of ionic strength on the energy of the transition state compared to the bound state due to larger distances between charged residues in the transition state. This model explains the faster dissociation rate at higher ionic strengths that may assist rapid turnover that is important for biological function. These results provide a quantitative model for coupling protein association with electron transfer and elucidate the role of short-range interactions in determining the rate of electron transfer.

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Ionic-strength-dependence of the oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by cytochrome c oxidase.

Project description:The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.

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Epitope mapping for the monoclonal antibody that inhibits intramolecular electron transfer in flavocytochrome b2.

Project description:Flavocytochrome b(2) (yeast L-lactate dehydrogenase) carries one FMN and one protohaem IX on each of its four subunits. The prosthetic groups are bound to separate domains, the haem domain (residues 1-99) and the flavin domain (residues 100-485), which interact for electron transfer between lactate-reduced FMN and haem b(2); in vivo, the latter reduces cytochrome c. In the crystal structure, one haem domain out of two is mobile. Previously we have described a monoclonal antibody, raised against the tetramer, that only recognizes the native haem domain and prevents electron transfer between flavin and haem, while having no effect on flavin reduction by the substrate [Miles, Lederer and Lê (1998) Biochemistry 37, 3440-3448]. In order to understand the structural basis of the uncoupling between the domains, we proceeded to site-directed mutagenesis, so as to map the epitope on the surface of the haem domain. We analysed the effects of 14 mutations at 12 different positions, located mostly in the domain interface or at its edge; we also analysed the effect of replacing protohaem IX with its dimethyl ester. We used as criteria the antibody-mediated inhibition of cytochrome c reduction by flavocytochrome b(2), competitive ELISA tests and surface plasmon resonance. We have thus defined a minimal epitope surface on the haem domain; it encompasses positions 63, 64, 65, 67, 69 and 70 and one or both haem propionates. When the haem and flavin domains are docked for electron transfer, the 65, 67 and 70 side chains, as well as the haem propionates, are excluded from solvent. The present results thus indicate that, when bound, the antibody acts as a wedge between the domains and constitutes a physical barrier to electron transfer.

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Isolation and characterization of the cytochrome domain of flavocytochrome b2 expressed independently in Escherichia coli.

Project description:The cytochrome domain of flavocytochrome b2 (L-lactate dehydrogenase) was expressed in the bacterium Escherichia coli and a purification procedure was developed. When expressed in E. coli, the b2-cytochrome domain contains protohaem IX and has an electronic absorption spectrum identical with that of the cytochrome b2 'core' produced by proteolytic cleavage of the enzyme isolated from yeast. The b2-cytochrome domain isolated from E. coli has an Mr of 10,500 and a redox potential of -31 +/- 2 mV. High-field n.m.r. studies indicate pKa values for the haem propionate groups to be 4.8 and 4.6, consistent with these groups being exposed to solvent rather than buried inside the protein. Using n.m.r. spectroscopy, we have determined an electron self-exchange rate constant for the b2-cytochrome domain of 2.3 x 10(6) M-1.s-1, which is more than two orders of magnitude larger than the value obtained for microsomal cytochrome b5, a homologue of b2-cytochrome domain.

| S-EPMC1130997 | biostudies-other

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

Project description:His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.

| S-EPMC2526232 | biostudies-literature

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| S-EPMC8339437 | biostudies-literature

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