Electron Transfer Kinetics between Hemoglobin Subunits

Laurent Kiger, Michael C. Marden
2001 Journal of Biological Chemistry  
The kinetics for electron transfer have been measured for samples of hemoglobin valency hybrids with initially one type of subunit, ␣ or ␤, in the oxidized state. Incubation of these samples under anaerobic conditions tends to randomize the type of subunit that is oxidized. With a time coefficient of a few hours at pH 7, 25°C, the Hb solution (0.1 mM heme) approaches a form with about 60% of ␤ chains reduced, indicating a faster transfer rate in the direction ␣ to ␤. There was no observable
more » ... s no observable electron transfer for samples saturated with oxygen. The electron transfer occurs predominantly between deoxy and aquo-met subunits, both high spin species. Furthermore, electron transfer does not depend on the quaternary state of hemoglobin. Incubation of oxidized crosslinked tetramer Hb A with deoxy Hb S also displayed electron transfer, implying a mechanism via inter-tetramer collisions. A dependence on the overall Hb concentration confirms this mechanism, although a small contribution of transfer between subunits of the same tetramer cannot be ruled out. These results suggest that in vivo collisions between the Hb tetramers will be involved in the relative distribution of the methemoglobin between subunits in association with the reductase system present in the erythrocyte. In biomolecules iron is in equilibrium between predominantly two oxidation states, the ferrous Fe(II) and the ferric Fe(III) states. In heme proteins such as Hb, the oxidation state will determine the ligands that bind to the iron of the active center heme; O 2 , CO, and NO bind to the ferrous form, whereas water, OH Ϫ , or CN Ϫ are ligands of the ferric state. Free heme in solution oxidizes on contact with oxygen, whereas the globin offers the possibility of reversible oxygen binding by prolonging the ferrous lifetime to more than 1 day. This is still low compared with the turnover of red blood cells of about 120 days, and reversal to the reduced state requires metHb 1 reductase. Although heme proteins can be generally classed as the electron transfer cytochromes and oxygen transporters (hemoglobin (Hb), myoglobin), the latter group obviously also participates in electron transfer reactions as part of their natural function. One of the great paradoxes of aerobic life is that although O 2 is essential for respiration, it also drives oxidation reactions, in particular that of the ferrous iron into its ferric form, giving rise to other oxidant species (O 2 . , H 2 O 2 , ROO ⅐ , OH ⅐ ). These free radicals, produced through the hemoglobin oxidation pathway, are linked to oxidative damage reactions (1, 2) such as the accumulation of heme degradation products in the membrane, which might accelerate the erythrocyte aging. Fortunately reducing systems inside the erythrocyte such as the NADH metHb reductase/cytochrome b 5 along with glutathione and ascorbic acid counterbalance the continuous in vivo autoxidation of hemoglobin, whereas catalase and superoxide dismutase, to cite the most familiar, participate in the elimination of the free radicals. If the oxygen binding properties and the autoxidation mechanism of hemoglobin are well described in the literature, the electron transfer reactions in hemoglobin intra-or inter-tetramers have not been extensively studied. In fact more is known about the electron transfer between Hb and other oxidant or reductant molecules. Furthermore, valency exchange in hemoglobin was often attributed to heme exchange between subunits; indeed, heme loss is relatively rapid for the oxidized or degraded form of hemoglobin. However, reduction of Hb by electron transfer is a natural process, and transfer between the subunits should also occur, although possibly on a much slower time scale. Electron transfer in hemoglobin and its redox-associated reactions are then linked to its in vivo life span. In the present work we study electron transfer reactions between hemoglobin molecules for a better understanding of this mechanism and the overall hemoglobin redox reactions that keep the oxygen carrier active in its ferrous form. We used simple methodologies to discriminate the bi-directional transfers ␣ 3 ␤ and ␤ 3 ␣ and intra versus inter-tetramer. Our results are discussed in light of Hb oxidation and reduction differences between the ␣ and ␤ subunit. MATERIALS AND METHODS Hb A was purified from the blood of healthy non-smoking donors, stripped of organophosphates, and stored under liquid nitrogen until use as described previously (3) . Hb S from homozygous sickle cell patients was purified on a DEAE-Sephadex column equilibrated with 50 mM Tris-HCl buffer, pH 8.3, after running a pH gradient 8.3 to 8.0. Natural variant Hb Chesapeake ␣92 Arg 3 Leu was found in a polyglobulic patient (4) and characterized by electrophoresis and molecular biology studies (DNA sequence analysis). The separation was accomplished by preparative isoelectric focalization as described below. MetHb was generated by autoxidation of ferrous Hb at 37°C overnight at pH 6.5 in 50 mM bis-Tris buffer with 100 mM NaCl. The remaining ferrous fraction was oxidized by a small excess of potassium ferricyanide at room temperature. The residual ferricyanide and its reduced form were removed by chromatography on an analytical grade ion exchange resin 501-X8 (Bio-Rad). Human ␣ and ␤ chains were split from Hb A upon reaction with p-mercuribenzoate after the procedure of Winterhalter and Colosimo (5) with some modifications. Then paramercuribenzoate chains were separated by preparative isoelectric focusing at 5°C using granulated gel (Ultrodex) and ampholines (with a pH gradient from 6 to 8; No. 80-1125-93) purchased from Amersham Biosciences, Inc. The SH groups were regenerated by incubation with dithiothreitol according to the method developed by Parkhurst and Parkhurst (6). Because the UV spectra are a sensitive probe of the presence of para-mercuribenzoate bound to the cysteines, the ␣SH and ␤SH spectra were recorded to assess the absence of residual paramercuribenzoate. Analytical isoelectric focusing in polyacrylamide
doi:10.1074/jbc.m106807200 pmid:11602592 fatcat:j3t3cntwwnbkxgfhbb7epe7qmi