Genetic Engineering of the Heme Pocket in Human Serum Albumin:Modulation of O2 Binding of Iron Protoporphyrin IX by Variation of Distal Amino Acids

摘要

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin(rHSA)generates an artificial hemoprotein,which can bind O2 in much the same way as hemoglobin(Hb).We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly:(i)Ile-142->His,which is axially coordinated to the central Fe~(2+)ion of the heme,and(ii)Tyr-161->Phe or Leu,which makes the sixth coordinate position available for ligand interactionsI142H/Y161F(HF)or I142H/Y161L(HL).Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids.We show that introduction of a third mutation on the distal side of the heme(at position Leu-185,Leu-182,or Arg-186)can modulate the O2 binding equilibrium.The coordination structures and ligand(O2 and CO)binding properties of nine rHSA(triple mutant)-heme complexes have been physjcochemically and kinetically characterized.Several substitutions were severely detrimental to O2 binding:for example,Gln-185,His-185,and His-182 all generated a weak six-coordinate heme,while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2.In marked contrast,HSA(HL/L185N)-heme showed very high O2 binding affinity(P_(1/2)~(O_2)1 Torr,22 deg C),which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb.Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant.Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment,yielded a complex with reduced O2 binding affinity(P_(1/2)~(O_2)9-10 Torr,22 deg C),which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.