Carboxyl Residues in the Iron−Sulfur Protein Are Involved in the Proton Pumping Activity of P. denitrificans bc1 Complex
| Autor: | Michele Lorusso, Marcus Korn, Sergio Papa, Giuseppe Cutecchia, Tiziana Cocco, Bernd Ludwig |
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| Rok vydání: | 2001 |
| Předmět: |
Iron-Sulfur Proteins
Macromolecular Substances Stereochemistry Protein subunit Molecular Sequence Data Biochemistry Electron Transport Complex III chemistry.chemical_compound Nucleophile Cytochrome C1 Amino Acid Sequence Fluorescein Paracoccus denitrificans Sequence Homology Amino Acid biology Cytochrome b Chemical modification Proton Pumps biology.organism_classification Fluorescence Dicyclohexylcarbodiimide chemistry Liposomes Quinolines |
| Zdroj: | Biochemistry. 40:15396-15402 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi011421b |
| Popis: | A study is presented on chemical modification of the three subunit Paracoccus denitrificans bc(1) complex. N-(Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment caused a loss of the proton pumping activity of liposome-reconstituted bc(1) complex. A similar effect, which is referred to as the decoupling effect, resulted upon reaction of N,N'-dicyclohexylcarbodiimide (DCCD) with the complex. Direct measurement of the binding of EEDQ to the complex subunits, performed in the presence of the fluorescent hydrophobic nucleophile 4'-[(aminoacetamido)methyl]fluorescein (AMF), showed that the iron-sulfur protein (ISP) and cytochrome c(1) were labeled by EEDQ, whereas cytochrome b was not. Tryptic digestion and sequencing analysis of the fluorescent fragment of the ISP revealed this to consist of a segment with six acidic residues, among which the highly conserved aspartate 160 is present. Analogous experiments on DCCD binding showed that all the three subunits of the complex were labeled. However, DCCD concentration dependence of carboxyl residue modification in the individual subunits and of proton pumping activity showed that the decrease of the H(+)/e(-) ratio correlated only with the modification of the ISP. Tryptic digestion of labeled ISP and sequencing analysis of the fluorescent fragment gave results superimposable upon those obtained with EEDQ. Chymotryptic digestion and sequencing analysis of the single fluorescent fragment of cytochrome b showed that this fragment contained glutamate 174 and aspartate 187. We conclude that, in the P. denitrificans bc(1) complex, carboxyl residues in cytochrome b do not appear to be critically involved in the proton pump mechanism of the complex. |
| Databáze: | OpenAIRE |
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