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Heme Pocket Architecture In Human Serum Albumin: Regulation Of O2 Binding Affinity Of A Prosthetic Heme Group By Site-Directed Mutagenesis

T. Komatsu, T. Komatsu, A. Nakagawa, E. Tsuchida
Published 2008 · Chemistry

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We present the O 2 binding properties of recombinant human serum albumin (rHSA) mutants complexed with an iron(ll) protoporphyrin IX as a prosthetic heme group. Iron(lll) 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 lle-i42; and (ii) the coordinated Tyr-161 was replaced with hydrophobic Leu using site-directed mutagenesis. It provided a recombinant HSA double-mutant [rHSA(l142H/ 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/Li85N)-heme showed high O 2 binding affinity (P O2 : 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 O2 ) to 10 Torr, which is almost equivalent to value for human red blood cells.
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