Site-directed mutagenesis of two conserved charged amino acids in the N-terminal region of alpha subunit of E. coli-F(0)F(1).
Two conserved charged amino acids of the N-terminal 'crown' region of the alpha subunit of E. coli-F(1), alpha-D36 and alpha-R40 were exchanged for chemically related (alpha-D36-->E, alpha-R40-->K) or unrelated amino acids (alpha D-36-->K, alpha R40-->G), respectively, by employing oligonucleotide-directed mutagenesis. ATP formation and ATP hydrolyzing activity of isolated plasma membrane vesicles was strongly inhibited in mutant HS2 (alpha-D36-->K), but only slightly affected in the other mutants. The inhibition is not due to a lower content of F0F1 in HS2. In this mutant the extent of the proton gradient generated by ATP hydrolysis was more than 80% inhibited; in all other transformants much smaller effects were observed. The proton gradient established by NADH oxidation was 33% decreased in HS2, but was decreased to a lesser extent in all other mutants. After blockage of F0 by DCCD treatment, the same NADH-induced proton gradient was obtained in all transformants including HS2. This and the fact that the activity of NADH oxidation was unchanged indicate increased proton leakiness of F0F1 carrying the alpha-D36-->K mutation. In F1 alpha-D36 is located in a domain contacting the beta subunit in the vicinity of the arginine beta-R52. The effect of alpha-D36-->K replacement on catalysis and coupling thus may be due to an electrostatic repulsive effect in the crown region which alters the alpha and beta interaction.
Hase
B
B
Institut für Biochemie der Pflanzen, Heinrich Heine Universität Düsseldorf, Germany.
Werner-Grüne
S
S
Deckers-Hebestreit
G
G
Strotmann
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000502:physiology*; D001224:Aspartic Acid / Q000502:physiology*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D017494:Proton Pumps / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017494:Proton Pumps; D009243:NAD; D001224:Aspartic Acid; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D001120:Arginine; D006180:Proton-Translocating ATPases