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

Abstract

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.

Evidence for pH-dependent multiple conformers in iron(II) heme–human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding

“…In agreement with previous results [34,38], the UV-vis spectrum of iron(II) heme-HSA at pH 7.0 is characterized by a broad Soret band centered at 418 nm, with two shoulders at 405 and 424 nm, and b and a bands at 536 and 572 nm, respectively ( . This latter form has been proposed to correspond to a 5cHS species containing a tyrosinate (possibly Tyr161) [14] coordinated to the iron(II) heme, whereas the formation of a 4cIS species is a consequence of the weakness of the heme Fe(II)-O Tyr bond [34].…”

“…Iron(III) heme is secured to HSA by the long IA-IB connecting loop that fits into the cleft opening [14-16, 29, 33, 34]. In turn, heme endows HSA with globin-like reactivity [4,[35][36][37][38][39] and spectroscopic properties [22, 30-32, 34, 40-43]. [14] HSA is crucial for heme scavenging, providing protection against free heme oxidative damage, limiting the access of pathogens to heme, and contributing to iron homeostasis by recycling the heme iron.…”

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

“…Interestingly, HSA-heme binds NO and CO and exhibits catalase and peroxidase activity [11,20,[24][25][26][27][28][29][30]. Furthermore, HSA-heme mutants have been proposed as O 2 -carriers [31,32]. Remarkably, abacavir modulates allosterically kinetics of peroxynitrite-mediated oxidation of human ferrous nitrosylated HSA-heme (HSA-heme-Fe(II)-NO) [33].…”

Serum heme‐albumin: An allosteric protein

“…Serum heme-albumin (SA-heme) displays heme-based reactivity ( (36,54). Remarkably, values of kinetic and thermodynamic parameters for binding of diatomic gaseous ligands to wild-type and engineered SA-heme-Fe(II) are similar to those reported for ferrous sperm whale Mb, generally taken as a molecular model of monomeric globins (36,54,61).…”

“…Because SA-heme mutants could be of relevant clinical importance not only as a red cell substitute but also as an O 2 -providing therapeutic reagent (54), the allosteric modulation of the heme reactivity by drugs should be investigated before the in vivo use of SA-heme-based blood substitutes.…”