Dynamics of 伪-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state

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• 作者：Laurent Kiger ; Corinne Vasseur ; Elisa Domingues-Hamdi ; Gilles Truan ; Michael C. Marden ; V茅ronique Baudin-Creuza
• 关键词：AHSP ; alpha hemoglobin-stabilizing protein ; AHSPWT ; recombinant human wild type AHSP with an N-terminal Gly-Pro-Leu-Gly-Ser peptide ; AHSP ; 伪-Hb ; complex formed between WT 伪-Hb chain and recombinant human wild type AHSP ; Cyt b5 ; recombinant soluble domain of human membrane-bound cytochrome b5 ; GST ; glutathione S-transferase ; Hb ; human adult hemoglobin ; MetHb ; oxidized Hb ; Ngb ; neuroglobin ; hemin ; ferric heme ; PBS ; phosphate-buffered saline ; RBCs ; red blood cells ; ROS ; reactive oxygen species
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：January, 2014
• 年：2014
• 卷：1840
• 期：1
• 页码：277-287
• 全文大小：1066 K

文摘

Background

AHSP is an erythroid molecular chaperone of the 伪-hemoglobin chains (伪-Hb). Upon AHSP binding, native ferric 伪-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).

Methods

Recombinant AHSP, WT 伪-Hb:AHSP and 伪-Hb^HE7Q:AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated 伪-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of 伪-Hb bound to AHSP as well as 伪-Hb release from the 伪-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.

Results

AHSP binding to 伪-Hb is kinetically controlled to prevail over direct binding with 尾-chains and is also thermodynamically controlled by the 伪-Hb redox state and not the liganded state of the ferrous 伪-Hb. The dramatic instability of isolated ferric 伪-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in 伪-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric 伪-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to 伪-Hb within the Hb-tetramer.

Conclusion

伪-Hb reduction could trigger its release from AHSP toward its final Hb 尾-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.

General significance

The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.