Spin label probes of the environment of cysteine beta-93 in hemoglobin.
The environment of cysteine beta-93 is altered during the oxygenation of hemoglobin. Electron spin resonance was used to probe the hemoglobin conformation in this crucial region on the proximal side of the heme. Spin-labeled hemoglobins in both the R-liganded state [methemoglobin and oxyhemoglobin] and the T-unliganded state [deoxyhemoglobin as well as Ni(II) and Cu(II) substituted hemoglobins] were investigated. Included in this study are iodoacetamide and maleimide labels with different constraints at the point of reaction with the SH-group, as well as a series of pyrrolidinyloxyl maleimide labels of different chain length. From differences in the correlation time of the spin labels it was possible to identify two distinct strongly immobilized configurations in addition to the relatively mobile configuration with the label on the surface of the protein. By dipolar interactions between the spin labels and paramagnetic Cu(II) at the heme center, the relative position of the three orientations for the spin label are defined. Differences are observed between the two hemoglobin conformations with respect to the relative population of the various orientations and with respect to the potential barrier associated with the reorientation of the spin labels.
Manoharan
P T
PT
Laboratory of Cellular and Molecular Biology, National Institutes of Health, National Institute on Aging, Gerontology Research Center, Baltimore, MD 21224.
Wang
J T
JT
Alston
K
K
Rifkind
J M
JM
D000818:Animals; D003300:Copper; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D005914:Globins / Q000648:ultrastructure*; D006454:Hemoglobins / Q000648:ultrastructure*; D006736:Horses / Q000097:blood; D006801:Humans; D008706:Methemoglobin / Q000648:ultrastructure; D015394:Molecular Structure; D009532:Nickel; D010108:Oxyhemoglobins / Q000648:ultrastructure; D011487:Protein Conformation; D013113:Spin Labels
D006454:Hemoglobins; D010108:Oxyhemoglobins; D013113:Spin Labels; D003300:Copper; D009532:Nickel; D005914:Globins; D008706:Methemoglobin; D003545:Cysteine