title: Subunit disassembly pathway of human hemoglobin revealing the site-specific role of its cysteine residues.
abstract: Cysteine residues play a unique role in human hemoglobin (Hb) by affecting its cooperative oxygen binding behavior and the stability of its tetrameric structure. However, how these cysteine residues fulfill their biophysical functions from the molecular level is yet unclear. Here we study the subunit disassembly pathway of human hemoglobin using the sulfhydryl reagent, p-hydroxymercuribenzoate (PMB) and investigate the functional roles of cysteine residues in human hemoglobin. We show evidence from the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that all three types of cysteine residues, including the surface-exposed βCys93 and the shielded αCys104 and βCys112 are reactive to PMB, resolving an issue long under debate. It is demonstrated that all three types of cysteine residues must be blocked by PMB to accomplish the subunit disassembly, and the PMB-cysteine reactions proceed in a stepwise manner with an order of βCys93, αCys104, and βCys112. The PMB reactions with the three different cysteine residues demonstrate strong site-specificity. The possible influence of PMB-cysteine reactions to the stability of various intersubunit salt bridges has been discussed based on the crystallographic structure of hemoglobin, providing insights in understanding the hemoglobin subunit disassembly pathway and the site-specific functional role of each cysteine residue in hemoglobin.
authors.affiliation: Department of Chemistry, National Sun Yat-sen University, Kaohsiung, Taiwan, R.O.C. 80424.
authors.forename: Heng-I
authors.initials: HI
authors.lastname: Kan
authors.forename: I-Ying
authors.initials: IY
authors.lastname: Chen
authors.forename: Muhammad
authors.initials: M
authors.lastname: Zulfajri
authors.forename: Chia C
authors.initials: CC
authors.lastname: Wang
mesh_terms: D003545:Cysteine / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006902:Hydroxymercuribenzoates / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006454:Hemoglobins; D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D003545:Cysteine