Heme Pocket Architecture in Human Serum Albumin: Regulation of O2 Binding Affinity of a Prosthetic Heme Group by Site-Directed Mutagenesis

Summary : We present the O₂ 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 O₂ 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 O₂ at room temperature. Laser flash photolysis experiments demonstrated that O₂ 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 O₂ binding affinity of rHSA(HL)–heme was considerably lower than those of R-state hemoglobin (Hb) and myoglobin (Mb), principally because of the high O₂ 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 O₂ binidng affinity. The rHSA(HL/L185N)–heme showed high O₂ binding affinity ( : 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 O₂ binding affinity ( ) to 10 Torr, which is almost equivalent to value for human red blood cells.