Reactivity of the N-terminal cysteine residues in active and inactive forms of FNR, and O2-responsive, Fe containing transcriptional regulator of Escherichia coli.
FNR, the O2-responsive gene regulator of anaerobic respiratory genes in Escherichia coli, contains an N-terminal cluster of four cysteine residues (Cys16-X3-Cys20-X2-Cys23-X5-Cys29), three of which are thought to be involved in the binding of an iron cofactor. The accessibility of the cysteine residues for iodoacetate is known to increase upon switch from the active (anaerobic) to the inactive (aerobic or metal depleted) state. It was analyzed which residues become accessible under either condition. Up to four modified forms, FNR-I, FNR-II, FNR-III, and FNR-IV, containing approximately 1, 2, 3.5, and 5 carboxymethyl groups, were obtained either by reaction in vivo and in vitro under conditions of aerobiosis, anaerobiosis, or iron limitation. By N-terminal sequencing, the carboxymethylated cysteine residues were identified. The amount of label in each of the four cysteine residues increased rather uniformly and gradually from FNR-I to FNR-IV irrespective of the condition of labeling; only Cys16 was preferentially labeled to some extent. It is concluded that the four essential cysteine residues change their accessibility in a similar way in the switch from active to inactive (aerobic or metal depleted) FNR, without specific differences in their reaction or function. Potential modes of Fe-binding by the cysteine residues are discussed. In addition, a different type of interaction of Fe(II) with FNR is described. The interaction occurred also in FNR carboxymethylated at approximately three cysteine residues.
Six
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Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, Germany.
Trageser
M
M
Kojro
E
E
Fahrenholz
F
F
Unden
G
G
D000332:Aerobiosis; D000478:Alkylation; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007461:Iodoacetates; D019807:Iodoacetic Acid; D007501:Iron / Q000032:analysis* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D014316:Tritium
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007461:Iodoacetates; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors; D014316:Tritium; D007501:Iron; D003545:Cysteine; D019807:Iodoacetic Acid