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

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

1992 ◽  
Vol 285 (1) ◽  
pp. 187-192 ◽  
Author(s):  
C S Miles ◽  
N Rouvière-Fourmy ◽  
F Lederer ◽  
F S Mathews ◽  
G A Reid ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Isotope Effects ◽  
Mutant Enzyme ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flavocytochrome B2 ◽  
Rate Determining Step ◽  
Kinetic Isotope

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.

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

1996 ◽  
Vol 316 (2) ◽  
pp. 507-513 ◽  
Author(s):  
R. Eryl SHARP ◽  
Stephen K. CHAPMAN ◽  
Graeme A. REID
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Lactate Dehydrogenase ◽  
Structural Integrity ◽  
Hinge Region ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Lactate Dehydrogenase Activity ◽  
Flavocytochrome B2

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.

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

2003 ◽  
Vol 373 (1) ◽  
pp. 115-123 ◽  
Author(s):  
K. H. Diêp LÊ ◽  
Martine MAYER ◽  
Florence LEDERER
Keyword(s):  
Monoclonal Antibody ◽  
Electron Transfer ◽  
Cytochrome C ◽  
Dimethyl Ester ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Intramolecular Electron Transfer ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

Flavocytochrome b2 (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 b2; 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 b2, 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.

STUDIES ON THE CYTOCHROME OXIDASE AND OXIDATION PATHWAY IN UREDOSPORES OF WHEAT STEM RUST

1961 ◽  
Vol 39 (5) ◽  
pp. 1131-1148 ◽  
Author(s):  
G. A. White ◽  
G. A. Ledingham
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Reductase Activity ◽  
Oxygen Affinity ◽  
Antimycin A ◽  
Respiratory Enzymes ◽  
Wheat Stem Rust ◽  
Malic Dehydrogenase ◽  
Cytochrome C Reductase

Electron transport to oxygen in a particulate fraction from uredospores of Puccinia graminis var. tritici occurs through a series of carriers similar to those of other fungi and higher plants.Experiments with various enzyme inhibitors and measurements of the oxygen affinity of respiration have shown that cytochrome oxidase mediates the final step in the sequence of electron transfer. The enzyme was localized in a fraction sedimenting at 20,000 g and was typically inhibited by cyanide, azide, and CO-dark, the latter inhibition being light-reversible. Other enzymes present were succinic-cytochrome c reductase, DPNH- and TPNH-cytochrome c reductase, dye reductase, malic dehydrogenase, isocitric dehydrogenase, and glycerol-1-phosphate dehydrogenase. Particulates failed to oxidize DPNH unless an electron acceptor was added. An increase in the activity of several of the respiratory enzymes was noted upon spore germination.Succinic-cytochrome c reductase was only partially sensitive to Antimycin A, HOQNO, and the naphthoquinone, SN 5949. These compounds markedly inhibited a labile portion of the DPNH-cytochrome c reductase activity but had little effect on the stable activity remaining in aged particles. Menadione, but not vitamin K1, stimulated electron transfer. Antimycin A and SN 5949 virtually blocked spore respiration suggesting a "Slater-type" factor in the intact pathway of oxidation.

A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect

2002 ◽  
Vol 362 (3) ◽  
pp. 749-754 ◽  
Author(s):  
Ziedulla Kh. ABDULLAEV ◽  
Marina E. BODROVA ◽  
Boris V. CHERNYAK ◽  
Dmitry A. DOLGIKH ◽  
Ruth M. KLUCK ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Antioxidant Activities ◽  
H2o2 Production ◽  
High Electron ◽  
Wild Type ◽  
Low Concentrations ◽  
Rat Heart Mitochondria ◽  
The Difference ◽  
Α Helix

A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal α-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H2O2 production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other ‘horse’ modifications in the N-terminal α-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2–12.

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