Refined Structure of Cytochromeb562fromEscherichia coliat 1.4 Å Resolution

Hamada, Kensaku; Bethge, P. H.; Mathews, Scott F.

doi:10.1006/jmbi.1995.0192

Cited by 73 publications

(85 citation statements)

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“…About 28% of the 14 C-ALA added to the medium was consumed during growth of the cells. The yield of 14 C-heme was 10 to 15%; i.e., about 50% of the consumed 14 C-ALA was recovered in the final 14 C-heme preparation. Eight ALA molecules are used to build up the heme molecule.…”

Section: Methodsmentioning

confidence: 99%

“…Eight ALA molecules are used to build up the heme molecule. The obtained radioactive heme has 14 C at positions 1, 3,6,8,11,13,17, and 19 of the tetrapyrrole molecule. Analysis of 14 C-heme preparations by reversed-phase high-pressure liquid chromatography (40) and thin-layer chromatography (9) showed only protoheme IX; no heme O or other porphyrin compounds were detected.…”

Section: Methodsmentioning

confidence: 99%

“…The acid acetone extracts were pooled and concentrated to 0.5 to 1 ml by evaporation. The yield of both 14 C-heme and nonlabeled heme with this procedure was typically about 20 nmol per 60-ml culture. About 28% of the 14 C-ALA added to the medium was consumed during growth of the cells.…”

Section: Methodsmentioning

confidence: 99%

“…The reason to use the mutant sdhC gene instead of the wild-type gene (which yields more cytochrome per cell) was that AN344, with wild-type sdhC on the plasmid, required high concentrations of ALA in the medium (Ͼ120 M) and also accumulated coproporphyrinogen III, an intermediate of the heme biosynthetic pathway. Strain AN344/pTYR13 was grown at 37°C in LB, supplemented with 12 M ferric citrate, 35 mg of ampicillin per liter, and 31 M (50.8 Ci/mol) [4-14 C]ALA ( 14 C-ALA; New England Nuclear) or 31 M ALA, for the production of 14 Cheme or nonlabeled heme, respectively. After overnight growth, the culture was harvested by centrifugation and washed twice in 30 mM Tris-HCl buffer, pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

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Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

1997

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Cytochromes of the c type contain covalently bound heme. In bacteria, they are located on the outside of the cytoplasmic membrane. Cytochrome c synthesis involves export of heme and apocytochrome across the cytoplasmic membrane followed by ligation of heme to the polypeptide. Using radioactive protoheme IX produced in Escherichia coli, we show that Bacillus subtilis can use heme from the growth medium for cytochrome c synthesis. The B. subtilis ccdA gene encodes a 26-kDa integral membrane protein which is required for cytochrome c synthesis (T. Schiött et al., J. Bacteriol. 179:1962-1973, 1997). In this work, we analyzed the stage at which cytochrome c synthesis is blocked in a ccdA deletion mutant. The following steps were found to be normal in the mutant: (i) transcription and translation of cytochrome c structural genes, (ii) translocation of apocytochrome across the cytoplasmic membrane, and (iii) heme transport from the cytoplasm to cytochrome polypeptide on the outer side of the cytoplasmic membrane. It is concluded that CcdA is required for a late step in the cytochrome c synthesis pathway.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

1997

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“…The protein has been well characterized both structurally (24,25) and biophysically (see, for example, refs. 26 and 27).…”

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confidence: 99%

Protein engineering of cytochrome b ₅₆₂ for quinone binding and light-induced electron transfer

Hay

Wallace

Smith

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The central photochemical reaction in photosystem II of green algae and plants and the reaction center of some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule. Through protein engineering, we have been able to modify a protein to mimic this reaction. A unique quinone-binding site was engineered into the Escherichia coli cytochrome b 562 by introducing a cysteine within the hydrophobic interior of the protein. Various quinones, such as p-benzoquinone and 2,3-dimethoxy-5-methyl-1,4-benzoquinone, were then covalently attached to the protein through a cysteine sulfur addition reaction to the quinone ring. The cysteine placement was designed to bind the quinone Ϸ10 Å from the edge of the bound porphyrin. Fluorescence measurements confirmed that the bound hydroquinone is incorporated toward the protein's hydrophobic interior and is partially solvent-shielded. The bound quinones remain redox-active and can be oxidized and rereduced in a two-electron process at neutral pH. The semiquinone can be generated at high pH by a one-electron reduction, and the midpoint potential of this can be adjusted by Ϸ500 mV by binding different quinones to the protein. The heme-binding site of the modified cytochrome was then reconstituted with the chlorophyll analogue zinc chlorin e 6. By using EPR and fast optical techniques, we show that, in the various chlorin-protein-quinone complexes, light-induced electron transfer can occur from the chlorin to the bound oxidized quinone but not the hydroquinone, with electron transfer rates in the order of 10 8 s ؊1 .photosynthetic reaction center ͉ artificial photosynthesis ͉ chlorophyll analog ͉ zinc chlorin ͉ cysteine A ll of the energy needs of plants and photoautotrophic bacteria are met by the light reactions of photosynthesis. The primary photochemistry involves the absorption of visible photons and the subsequent conversion of the absorbed energy into chemical potential energy by the formation of a chargeseparated state. This photochemistry takes place in membranebound protein complexes called reaction centers (RCs). The best understood of these systems are the bacterial RCs from purple and green filamentous bacteria. The primary photochemistry that occurs in the bacterial RC is also common to the RC in photosystem II of cyanobacteria and higher plants (reviewed in refs. 1-3). The photochemical reactions in the RCs occur through the transfer of an electron from a light-excited (bacterio)chlorophyll [(B)Chl] through the protein, by means of a (bacterio)pheophytin, to a bound quinone molecule. The cofactors are Ϸ10 Å apart so that the electron transfer (ET) reaction is nonadiabatic, occurring through a quantum tunneling mechanism (4-6). This ET reaction has been mimicked by using organic ''diad'' molecules consisting of a covalently linked, light-activated electron donor, such as a porphyrin, and an electron acceptor, such as a quinone, for several decades with considerable success (7-10).The protein environment play...

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b‐Type Cytochrome Electron Carriers: Cytochromesb₅₆₂andb₅, and Flavocytochromeb₂

Mathews¹

2004

Handbook of Metalloproteins

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b‐Type cytochromes form a class of proteins that contain a heme (iron‐protoporphorin IX) bound non‐covalently to its polypeptide chain with two of its side chains coordinated to the iron. One of these side chains is always histidine and the other can be histidine or methionine. Cytochromes b 5 and b 562 are simple mono‐domain proteins that contain heme as the only prosthetic group and histidine and methionine, respectively, as the second heme ligand. Flavocytochrome b 2 also contains histidine as the second heme ligand, but contains a second prosthetic group, flavin mononucleotide (FMN), that resides in a separate domain. Cytochromes b 5 and b 562 each mediate intracellular electron transfer between redox partners, the former within the endoplasmic reticulum of mammalian cells and the latter within bacteria. Flavocytochrome b 2 catalyzes the oxidation of lactic acid within yeast mitochondria, with the cytochrome domain mediating electron transfer from substrate‐reduced FMN to exogenous cytochrome c. The structural, electrochemical, and catalytic properties of these three proteins are presented, along with the results of mutagenesis.

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Refined Structure of Cytochromeb562fromEscherichia coliat 1.4 Å Resolution

Cited by 73 publications

References 0 publications

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Protein engineering of cytochrome b ₅₆₂ for quinone binding and light-induced electron transfer

b‐Type Cytochrome Electron Carriers: Cytochromesb₅₆₂andb₅, and Flavocytochromeb₂

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Refined Structure of Cytochromeb562fromEscherichia coliat 1.4 Å Resolution

Cited by 73 publications

References 0 publications

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Protein engineering of cytochrome b 562 for quinone binding and light-induced electron transfer

b‐Type Cytochrome Electron Carriers: Cytochromesb562andb5, and Flavocytochromeb2

Contact Info

Product

Resources

About

Protein engineering of cytochrome b ₅₆₂ for quinone binding and light-induced electron transfer

b‐Type Cytochrome Electron Carriers: Cytochromesb₅₆₂andb₅, and Flavocytochromeb₂