Premium
Heme Pocket Architecture in Human Serum Albumin: Regulation of O 2 Binding Affinity of a Prosthetic Heme Group by Site‐Directed Mutagenesis
Author(s) -
Komatsu Teruyuki,
Nakagawa Akito,
Tsuchida Eishun
Publication year - 2008
Publication title -
macromolecular symposia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.257
H-Index - 76
eISSN - 1521-3900
pISSN - 1022-1360
DOI - 10.1002/masy.200851022
Subject(s) - heme , chemistry , hemeprotein , human serum albumin , histidine , porphyrin , myoglobin , stereochemistry , protoporphyrin ix , hemoglobin , hemin , protoporphyrin , mutagenesis , mutant , biochemistry , amino acid , organic chemistry , photodynamic therapy , gene , enzyme
Summary : We present the O 2 binding properties of recombinant human serum albumin (rHSA) mutants complexed with an iron(II) protoporphyrin IX as a prosthetic heme group. Iron(III) protoporphyrin IX (hemin) is bound within subdomain IB of HSA with weak axial coordination by Tyr‐161. In order to confer O 2 binding capability to this naturally occurring hemoprotein: (i) a proximal histidine was introduced into position Ile‐142; and (ii) the coordinated Tyr‐161 was replaced with hydrophobic Leu using site‐directed mutagenesis. It provided a recombinant HSA double‐mutant [rHSA(I142H/Y161L) = rHSA(HL)]. The rHSA(HL)–heme formed a ferrous five‐coordinate high‐spin complex with axial ligation of His‐142 under an Ar atmosphere. This artificial hemoprotein binds O 2 at room temperature. Laser flash photolysis experiments demonstrated that O 2 rebinidng to rHSA(HL)–heme displays monophasic kinetics, whereas the CO recombination process obeyed a double‐exponential pattern. This might be attributable to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O 2 binding affinity of rHSA(HL)–heme was considerably lower than those of R‐state hemoglobin (Hb) and myoglobin (Mb), principally because of the high O 2 dissociation rate constant. The third mutations have been introduced into the distal side of the heme (at position Leu‐185 or Arg‐186) to increase the O 2 binidng affinity. The rHSA(HL/L185N)–heme showed high O 2 binding affinity ( $P_{1/2}^{{\rm O}_2 } $ : 1 Torr), which is 18‐fold greater than that of the original double mutant rHSA(HL)–heme and which is rather close to those of Hb (R‐state) and Mb. Furthermore, replacement of polar Arg‐186 with Leu or Phe adjusted the O 2 binding affinity ( $P_{1/2}^{{\rm O}_2 } $ ) to 10 Torr, which is almost equivalent to value for human red blood cells.