Replacement of the heme axial lysine as a test of conformational adaptability in the truncated hemoglobin THB1.

Amino acid replacement is a useful strategy to assess the roles of axial heme ligands in the function of native heme proteins. THB1, the protein product of the Chlamydomonas reinhardtii THB1 gene, is a group 1 truncated hemoglobin that uses a lysine residue in the E helix (Lys53, at position E10 by reference to myoglobin) as an iron ligand at neutral pH. Phylogenetic evidence shows that many homologous proteins have a histidine, methionine or arginine at the same position. In THB1, these amino acids would each be expected to convey distinct reactive properties if replacing the native lysine as an axial ligand. To explore the ability of the group 1 truncated Hb fold to support alternative ligation schemes and distal pocket conformations, the properties of the THB1 variants K53A as a control, K53H, K53M, and K53R were investigated by electronic absorption, EPR, and NMR spectroscopies. We found that His53 is capable of heme ligation in both the Fe(III) and Fe(II) states, that Met53 can coordinate only in the Fe(II) state, and that Arg53 stabilizes a hydroxide ligand in the Fe(III) state. The data illustrate that the group 1 truncated Hb fold can tolerate diverse rearrangement of the heme environment and has a strong tendency to use two protein side chains as iron ligands despite accompanying structural perturbations. Access to various redox pairs and different responses to pH make this protein an excellent test case for energetic and dynamic studies of heme ligation.