Genetic Engineering of the Heme Pocket in Human Serum Albumin: Modulation of O2 Binding of Iron Protoporphyrin Ix by Variation of Distal Amino Acids.
From: Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan. teruyuki@waseda.jp
Journal of the American Chemical Society
- Publish Date: Sep 2007
- ISSN: 0002-7863
- Volume: 129
- Issue: 36
- Pages: 11286-95
- Medium: Print
- Language: English
- Citation (JAMA): Komatsu Teruyuki, Nakagawa Akito, Zunszain Patricia A, et al. Genetic Engineering of the Heme Pocket in Human Serum Albumin: Modulation of O2 Binding of Iron Protoporphyrin Ix by Variation of Distal Amino Acids.. J. Am. Chem. Soc. Sep 2007;129:11286-95
Abstract
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 Fe2+ ion of the heme, and (ii) Tyr-161 — >Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/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 physicochemically 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 (P1/2O2 1 Torr, 22 degrees 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 (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.
Mesh Headings (Keywords): Amino Acid Sequence, Binding Sites, Genetic Engineering, Heme, Humans, Models, Molecular, Oxygen, Protein Binding, Protein Conformation, Protoporphyrins, Serum Albumin
Check for Full Text / PubMed Unique Identifier (PMID): 17705494
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