Dioxygenation of Human Serum Albumin Having a Prosthetic Heme Group in a Tailor-Made Heme Pocket

Human serum albumin (HSA) is the most abundant plasma protein in our bloodstream and serves as a transporter for small hydrophobic molecules such as fatty acids, bilirubin, and steroids. Hemin dissociated from methemoglobin is also bound within a narrow D-shaped cavity in subdomain IB of HSA. In terms of the general hydrophobicity of the α-helical pocket, HSA potentially has features similar to the heme-binding site of myoglobin (Mb) or hemoglobin (Hb). However, the reduced ferrous HSA−heme complex is immediately oxidized by O2, because HSA lacks the proximal histidine that enables the heme group to bind O2. In this paper, we report the introduction of a proximal histidine into the subdomain IB of HSA by site-directed mutagenesis to construct a tailor-made heme pocket (I142H/Y161L), which allows a reversible O2 binding to the prosthetic heme group. Laser flash photolysis experiments revealed that this artificial hemoprotein appears to have two different geometries of the axial-imidazole coordination, and these two species (I and II) showed rather low O2 binding affinities (P1/2O2 = 18 and 134 Torr) relative to those of Mb and Hb

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

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 °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 °C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport

Photosensitized Reduction of Water to Hydrogen Using Human Serum Albumin Complexed with Zinc−Protoporphyrin IX

We present the photophysical properties of complexes of recombinant human serum albumin (rHSA) with Zn(II)−protoporphyrin IX (ZnPP) and their activities in the photosensitized reduction of water to hydrogen (H2) using methyl viologen (MV2+) as an electron relay. The ZnPP is bound in subdomain IB of wild-type rHSA [rHSA(wt)] by an axial coordination of Tyr-161 and, in the rHSA(I142H/Y161L) mutant [rHSA(His)], by a His-142 coordination. Both the rHSA(wt)−ZnPP and rHSA(His)−ZnPP complexes showed a long-lived photoexcited triplet state with lifetimes (τT) of 11 and 2.5 ms, respectively. The accommodation of ZnPP into the protein matrix efficiently eliminated the collisional triplet self-quenching process. The addition of a water-soluble electron acceptor, MV2+, resulted in a significant decrease in the triplet lifetime. The transition absorption spectrum revealed the oxidative quenching of rHSA−3ZnPP* by MV2+. The quenching rate constant (kq) and backward electron transfer rate constant (kb) were determined to be 1.4 × 107 and 4.7 × 108 M-1 s-1 for rHSA(wt)−ZnPP. In the presence of the colloidal PVA−Pt as a catalyst and triethanolamine (TEOA) as a sacrificial electron donor, the photosensitized reduction of water to H2 takes place. The efficiency of the photoproduction of H2 was greater than that of the system using the well-known organic chromophore, tetrakis(1-methylpyridinium-4-yl)porphinatozinc(II) (ZnTMPyP4+), under the same conditions

O₂ and CO Binding Properties of Artificial Hemoproteins Formed by Complexing Iron Protoporphyrin IX with Human Serum Albumin Mutants

The binding properties of O2 and CO to recombinant human serum albumin (rHSA) mutants with a prosthetic heme group have been physicochemically and kinetically characterized. Iron(III) protoporphyrin IX (hemin) is bound in subdomain IB of wild-type rHSA [rHSA(wt)] with weak axial coordination by Tyr-161. The reduced ferrous rHSA(wt)−heme under an Ar atmosphere exists in an unusual mixture of four- and five-coordinate complexes and is immediately autoxidized by O2. To confer O2 binding capability on this naturally occurring hemoprotein, a proximal histidine was introduced into position Ile-142 or Leu-185 by site-directed mutagenesis. A single mutant (I142H) and three double mutants (I142H/Y161L, I142H/Y161F, and Y161L/L185H) were prepared. Both rHSA(I142H/Y161L)−heme and rHSA(I142H/Y161F)−heme formed ferrous five-N-coordinate high-spin complexes with axial ligation of His-142 under an Ar atmosphere. These artificial hemoproteins bind O2 at room temperature. Mutation at the other side of the porphyrin, Y161L/L185H, also allowed O2 binding to the heme. In contrast, the single mutant rHSA(I142H)−heme could not bind O2, suggesting that removal of Y161 is necessary to confer reversible O2 binding. Laser flash photolysis experiments showed that the kinetics of CO recombination with the rHSA(mutant)−heme were biphasic, whereas O2 rebinding exhibited monophasic kinetics. This could be due 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 affinities of the rHSA(mutant)−heme were significantly lower than those of hemoglobin and myoglobin, principally due to the high O2 dissociation rates. Changing Leu-161 to Phe-161 at the distal side increased the association rates of both O2 and CO, which resulted in enhanced binding affinity