The Molecular Mechanism of Autoxidation for Human Oxyhemoglobin

Abstract

Human oxyhemoglobin showed a biphasic autoxidation curve containing two rate constants, i.e. k f for the fast autoxidation due to the ␣ chains, and k s for the slow autoxidation of the ␤ chains, respectively. Consequently, the autoxidation of the HbO 2 tetramer produces two different curves from the pH dependence of k f and k s . The analysis of these curves revealed that the ␤ chain of the HbO 2 tetramer does not exhibit any protoncatalyzed autoxidation, unlike the ␣ chain, where a proton-catalyzed process involving the distal histidine residue can play a dominant role in the autoxidation rate. When the ␣ and ␤ chains were separated from the HbO 2 tetramer, however, each chain was oxidized much more rapidly than in the tetrameric parent. Moreover, the separated ␤ chain was recovered completely to strong acid catalysis in its autoxidation rate. These new findings lead us to conclude that the formation of the ␣ 1 ␤ 1 contact produces in the ␤ chain a conformational constraint whereby the distal histidine at position 63 is tilted away slightly from the bound dioxygen, preventing the proton-catalyzed displacement of O 2. by a solvent water molecule. The ␤ chains have thus acquired a delayed autoxidation in the HbO 2 tetramer.The reversible and stable binding of molecular oxygen to the heme iron(II) is the basis of hemoglobin function. Consequently, much attention has been directed to the elucidation of the molecular mechanism of cooperative oxygen binding to the hemoglobin tetramer (1). However, the oxygenated form of hemoglobin, as well as of myoglobin, is known to be oxidized easily to the ferric(III) met form, which cannot bind molecular oxygen and is therefore physiologically inactive, with generation of the superoxide anion (2-5).To this autoxidation reaction, it has been widely accepted that hemoglobin is much more resistant as compared with myoglobin. Unlike myoglobin, Mansouri and Winterhalter (6) reported that oxyhemoglobin (HbO 2 ) showed a biphasic autoxidation reaction with a fast and a slow component. They also demonstrated that the ␣ chain was oxidized more rapidly than the ␤ chain in hemoglobin tetramer. At the same time, however, there have been a number of reports that such a rate difference was not observed between the ␣ and ␤ chains in the autoxidation reaction of HbA (7-9). Rather, Zhang et al. (9) showed that the rate of autoxidation was markedly enhanced when the HbO 2 tetramer dissociates into ␣␤ dimers. To clarify these discrepancies, we have recently examined systematically the effect of hemoglobin concentration on the autoxidation rate at several different values of pH, and found that human HbO 2 exhibits a biphasic autoxidation curve only in the pH range from neutral to acidic (5). By dissociation of tetramers into ␣␤ dimers, the rate of autoxidation for the fast component (due to the ␣ chain) was also found to increase markedly at the acidic pH, but the addition of 2,3-diphosphoglyceric acid offered no significant effect on the increment of the autoxidation rate (5).In the present pape...

Hemes férricos pentacoordenados e hexacoordenados dos monômeros d nativo e reconstituído da hemoglobina extracelular de Glossoscolex paulistus: estudos espectroscópicos em meio ácido

“…Nos dois conjuntos de espectros, observa-se que em pH 7,0, as bandas de Soret estão centradas em 413 nm e as bandas Q (α e β) possuem um máximo em 565 e 535 nm, sendo estas, características de hemicromo 30,31,[33][34][35] . Com a acidificação, observa-se tanto uma diminuição da intensidade como um deslocamento hipsocrômico da banda de Soret para 371 nm, juntamente com um deslocamento batocrômico da banda de transferência de carga do ligante para o metal (TCLM) para 643 nm.…”

Biochemical Fates of α Hemoglobin Bound to α Hemoglobin-stabilizing Protein AHSP

“…The rate of auto-oxidation of free oxy-␣Hb is roughly equal to that of Hb dimers, ϳ0.001 min Ϫ1 (16). The dependence of the observed rates of auto-oxidation on total ␣Hb (P 0 ) were fitted to Equations 1 and 2 (Fig.…”

Estudo espectroscópico do equilíbrio entre espécies hexacoordenada e pentacoordenada dos monômero d nativo e reconstituído da hemoglobina extracelular gigante de Glossoscolex paulistus em meio alcalino

“…Em pH 7,0, para ambos os casos, a banda de Soret está centrada em 413 nm e as bandas β e α possuem máximos, respectivamente, em 535 e 565 nm, constituindo, por conseguinte, espectros característicos de hemicromo 34,[45][46][47][48] . Isto indica que o método de separação cromatográfica em pH 9,0 gerou as condições para a transição da espécie aquometa para a espécie hemicromo, que consiste em um complexo bis-histidina.…”

“…Estudos envolvendo auto-oxidação mostram que, à temperatura ambiente, a conversão de uma espécie hidroxi-meta em hemicromo é muito rápida 48 . Estudos de auto-oxidação em meio alcalino envolvendo a hemoglobina G. paulistus corroboram tal assertiva 70 , ou seja, mesmo que ocorra a formação da espécie hidroxi-meta no meio em questão, esta espécie rapidamente se transformaria na espécie hemicromo, inviabilizando sua detecção, nos moldes em que o presente estudo foi desenvolvido, uma vez que o processo de separação cromatográfica já houvera proporcionado a transição aquometa→hemicromo.…”