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 in Rhizobium meliloti FixL, 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 from fixL recombinant Escherichia coli revealed 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.
Nakamura
H
H
The Institute of Physical and Chemical Research (RIKEN), Wako, 351-01, Japan. hironaka@postman.riken.go.jp
Saito
K
K
Ito
E
E
Tamura
K
K
Tsuchiya
T
T
Nishigaki
K
K
Shiro
Y
Y
Iizuka
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000071677:Histidine Kinase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006420:Hemeproteins; D011994:Recombinant Proteins; D006418:Heme; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen