Identification of the Hydrophobic Amino Acid Residues Required for Heme Assembly in the Rhizobial Oxygen Sensor Protein FixL

Rhizobial FixL is a novel heme protein, which senses environmental oxygen tension and directs signal transduction via protein phosphotransfer. To identify the essential residues for heme assembly inRhizobium melilotiFixL, we individually replaced the 18 invariant hydrophobic amino acid residues (F,...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 1998-06, Vol.247 (2), p.427-431
Main Authors: Nakamura, Hiro, Saito, Ken, Ito, Eiichi, Tamura, Koji, Tsuchiya, Terumasa, Nishigaki, Koichi, Shiro, Yoshitsugu, Iizuka, Tetsutaro
Format: Article
Language:English
Subjects:
ACIDE AMINE
Amino Acid Sequence
AMINO ACIDS
AMINOACIDOS
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Base Sequence
Binding Sites - genetics
DNA, Bacterial - genetics
Escherichia coli
Escherichia coli - genetics
Heme - chemistry
Hemeproteins - chemistry
Hemeproteins - genetics
Hemeproteins - metabolism
IDENTIFICACION
IDENTIFICATION
Kinetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Oxidation-Reduction
Oxygen - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
RHIZOBIUM MELILOTI
Signal Transduction
Sinorhizobium meliloti - genetics
Sinorhizobium meliloti - metabolism
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Summary:Rhizobial FixL is a novel heme protein, which senses environmental oxygen tension and directs signal transduction via protein phosphotransfer. To identify the essential residues for heme assembly inRhizobium melilotiFixL, we individually replaced the 18 invariant hydrophobic amino acid residues (F, I, L, and V) in the heme-containing domain with alanine and histidine. Spectroscopic measurements of the soluble fractions fromfixLrecombinantEscherichia colirevealed that V152, F162, F170, I172, L185, F226, L230, and F243 as well as the proximal ligand H194 were indispensable for heme assembly. Autoxidation rates of purified I209H, I210A, and I210H were 65-fold, 15-fold, and 15-fold, respectively, faster than that of the wild type, although they retained heme in the protein. The absorption peak in the Soret region of the ferric I209H or I210H was red-shifted, suggesting that the ferric heme is a hexa-coordinate form in these mutants.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1998.8694