Komatsu Teruyuki, Nakagawa Akito, Zunszain Patricia A, Curry Stephen, Tsuchida Eishun
Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
J Am Chem Soc. 2007 Sep 12;129(36):11286-95. doi: 10.1021/ja074179q. Epub 2007 Aug 18.
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.
将铁原卟啉IX嵌入重组人血清白蛋白（rHSA）经基因工程改造的血红素口袋中，可生成一种人工血红蛋白，其结合氧气的方式与血红蛋白（Hb）极为相似。我们之前证明了两个使辅基血红素基团能够可逆结合氧气所必需的突变：（i）Ile-142→His，它与血红素的中心Fe2+离子轴向配位；（ii）Tyr-161→Phe或Leu，这使得第六配位位置可用于配体相互作用[I142H/Y161F（HF）或I142H/Y161L（HL）]。在此，我们描述了另外一些新的突变，这些突变旨在通过特异性改变远端氨基酸来操纵rHSA-血红素复合物中血红素口袋的结构。我们发现，在血红素远端（Leu-185、Leu-182或Arg-186位置）引入第三个突变可调节氧气结合平衡。已对9种rHSA（三重突变体）-血红素复合物的配位结构和配体（O2和CO）结合特性进行了物理化学和动力学表征。几种取代对氧气结合极为不利：例如，Gln-185、His-185和His-182均产生一种弱六配位血红素，而rHSA（HF/R186H）-血红素复合物具有典型的双组氨酸血色素，会立即被氧气自动氧化。与之形成鲜明对比的是，HSA（HL/L185N）-血红素表现出非常高的氧气结合亲和力（P1/2O2为1 Torr，22℃），比原始双突变体rHSA（HL）-血红素高18倍，且非常接近肌红蛋白和高亲和力形式的Hb所表现出的亲和力。在185位引入Asn主要通过降低氧气解离速率常数来增强氧气结合。用Leu或Phe取代极性的Arg-186增加了远端环境的疏水性，产生一种氧气结合亲和力降低的复合物（P1/2O2为9 - 10 Torr，22℃），不过这与人类红细胞的亲和力几乎相同，因此更适合在氧气运输中发挥作用。
