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 oxy gen tension and directs signal transduction via protein phosphotransfe r, To identify the essential residues for heme assembly in Rhizobium m eliloti FixL, we individually replaced the 18 invariant hydrophobic am ino acid residues (F, I, L, and V) in the heme-containing domain with alanine and histidine. Spectroscopic measurements of the soluble fract ions 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 purif ied I209H, I210A, and I210H were 65-fold, 15-fold, and 15-fold, respec tively, 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 redshifted, suggesting that the ferric heme is a h exa-coordinate form in these mutants. (C) 1998 Academic Press.