Recombinant Hemoglobin(29Leucine Phenylalanine, 
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- 作者：Seong Tae Jeong ; Nancy T. Ho ; Michael P. Hendrich ; and Chien Ho
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：October 5, 1999
- 年：1999
- 卷：38
- 期：40
- 页码：13433 - 13442
- 全文大小：120K
- 年卷期：v.38,no.40(October 5, 1999)
- ISSN：1520-4995
文摘
Using our hemoglobin expression system in Escherichia coli, we have constructed threerecombinant hemoglobins (rHbs) with amino acid substitutions located in the 
gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">1 and gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">2 subunitinterfaces and in the distal heme pocket of the gifchars/alpha.gif" BORDER=0>-chain: rHb(gifchars/alpha.gif" BORDER=0>V96W, gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K), rHb(gifchars/alpha.gif" BORDER=0>L29F, gifchars/alpha.gif" BORDER=0>V96W,gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K), and rHb(gifchars/alpha.gif" BORDER=0>L29F). rHb(gifchars/alpha.gif" BORDER=0>V96W, gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K) exhibits low oxygen affinity and high cooperativityand also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has beenreported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley etal. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g., Leu
gif"> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutationinto our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(gifchars/alpha.gif" BORDER=0>L29F, gifchars/alpha.gif" BORDER=0>V96W,gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant,rHb(gifchars/alpha.gif" BORDER=0>V96W, gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K), but still exhibits low oxygen affinity and good cooperativity. According to electronparamagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionicform of bishistidine hemichrome. Previous reports have suggested that this form does not have waterpresent at the distal heme pocket. 1H nuclear magnetic resonance spectra of the triple mutant in the ferricstate also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a seriesof biochemical measurements to characterize these three interesting rHbs and to compare them to humannormal adult hemoglobin. These results provide new insights into the structure-function relationship ofhemoglobin with amino acid substitutions in the gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">1 and gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">2 interfaces and in the heme pockets.
gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">1 and gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">2 subunitinterfaces and in the distal heme pocket of the gifchars/alpha.gif" BORDER=0>-chain: rHb(gifchars/alpha.gif" BORDER=0>V96W, gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K), rHb(gifchars/alpha.gif" BORDER=0>L29F, gifchars/alpha.gif" BORDER=0>V96W,gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K), and rHb(gifchars/alpha.gif" BORDER=0>L29F). rHb(gifchars/alpha.gif" BORDER=0>V96W, gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K) exhibits low oxygen affinity and high cooperativityand also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has beenreported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley etal. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g., Leugif"> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutationinto our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(gifchars/alpha.gif" BORDER=0>L29F, gifchars/alpha.gif" BORDER=0>V96W,gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant,rHb(gifchars/alpha.gif" BORDER=0>V96W, gifchars/beta2.gif" BORDER=0 ALIGN="middle">N108K), but still exhibits low oxygen affinity and good cooperativity. According to electronparamagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionicform of bishistidine hemichrome. Previous reports have suggested that this form does not have waterpresent at the distal heme pocket. 1H nuclear magnetic resonance spectra of the triple mutant in the ferricstate also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a seriesof biochemical measurements to characterize these three interesting rHbs and to compare them to humannormal adult hemoglobin. These results provide new insights into the structure-function relationship ofhemoglobin with amino acid substitutions in the gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">1 and gifchars/alpha.gif" BORDER=0>1gifchars/beta2.gif" BORDER=0 ALIGN="middle">2 interfaces and in the heme pockets.