Abstract
Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.
Highlights
Hemoglobin (Hb)1 functions as an oxygen carrier only when reduced and is so maintained in vivo through enzymatic NADPH-driven reduction reactions [1,2,3,4]
We owe much of our understanding of copper-induced oxidation of Hb to the work of Winterbourn and Rifkind and coworkers [1, 5], who showed that copper-induced oxidation of oxygenated Hb (oxyHb) occurs through an internal electron transfer pathway between the iron atoms of the  chain hemes and copper bound at or near the 93 sulfhydryls
We have used Hb Deer Lodge and Hb A0 in the present studies in which we show that CO/H2O-driven electron transfer can bring about a reduction of the  chain hemes that utilizes this internal electron transfer pathway
Summary
Samples of native adult human hemoglobin (Hb A0) and Hb Deer Lodge were prepared by the ammonium sulfate method, chromatographically purified, and stripped of organic phosphate cofactors as described previously [7]. Regeneration of the SH groups of the  chains was done by treatment with -mercaptoethanol to reduce SH groups, followed by chromatography through DEAE-cellulose to remove para-hydroxymercuribenzoate. Elemental analysis of samples by atomic absorption spectroscopy (Perkin-Elmer 5000) following copper treatment validated the copper:iron ratios used and demonstrated that our Chelex chromatography of samples treated with a 5-fold excess of copper typically reduced the copper:heme ratio to about 0.6:1, consistent with retention of copper at unreactive high affinity sites and partial retention of copper at the lower affinity sites associated with electron transfer to the heme. Prolonged exposure to Chelex eventually removes all Cu. Incubations under CO were done with chemically pure grade (Յ99.5% purity) CO gas provided by National Welders, Inc. For visibleregion spectroscopy of copper-Hb complexes, CO gas was added to tonometers containing thoroughly deoxygenated metHb samples. Spin concentrations were determined by double integration using Cu(II)-EDTA, equine ferricytochrome c, and a quantitatively high spin methemoglobin sample as integration standards for the g ϭ 2, 3, and 6 regions of spectra, respectively
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