Heme Structures of Five Variants of Hemoglobin M Probed by Resonance Raman Spectroscopy

Abstract

The alpha-abnormal hemoglobin (Hb) M variants show physiological properties different from the beta-abnormal Hb M variants, that is, extremely low oxygen affinity of the normal subunit and extraordinary resistance to both enzymatic and chemical reduction of the abnormal met-subunit. To get insight into the contribution of heme structures to these differences among Hb M's, we examined the 406.7-nm excited resonance Raman (RR) spectra of five Hb M's in the frequency region from 1700 to 200 cm(-1). In the high-frequency region, profound differences between met-alpha and met-beta abnormal subunits were observed for the in-plane skeletal modes (the nu(C=C), nu(37), nu(2), nu(11), and nu(38) bands), probably reflecting different distortions of heme structure caused by the out-of-plane displacement of the heme iron due to tyrosine coordination. Below 900 cm(-1), Hb M Iwate [alpha(F8)His --> Tyr] exhibited a distinct spectral pattern for nu(15), gamma(11), delta(C(beta)C(a)C(b))(2,4), and delta(C(beta)C(c)C(d))(6,7) compared to that of Hb M Boston [alpha(E7)His --> Tyr], although both heme irons are coordinated by Tyr. The beta-abnormal Hb M variants, namely, Hb M Hyde Park [beta(F8)His --> Tyr], Hb M Saskatoon [beta(E7)His --> Tyr], and Hb M Milwaukee [beta(E11)Val --> Glu], displayed RR band patterns similar to that of metHb A, but with some minor individual differences. The RR bands characteristic of the met-subunits of Hb M's totally disappeared by chemical reduction, and the ferrous heme of abnormal subunits was no longer bonded with Tyr or Glu. They were bonded to the distal (E7) or proximal (F8) His, and this was confirmed by the presence of the nu(Fe-His) mode at 215 cm(-1) in the 441.6-nm excited RR spectra. A possible involvement of heme distortion in differences of reducibility of abnormal subunits and oxygen affinity of normal subunits is discussed.

Deciphering the Structural Role of Histidine 83 for Heme Binding in Hemophore HasA

“…The 3 band observed at 1477 cm Ϫ1 is consistent with a 6c HS heme. The second 3 band at 1487 cm Ϫ1 falls at the low end of the frequency range expected for 5c HS proteins (29). The more intense 2 band is consistent with the presence of a 6c LS species, although no 3 band attributable to 6c LS heme can be observed in the Soret-excited spectra recorded at pH 5.2 and 7.2.…”

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

“…Although vinyl modes with a comparable low frequency have been observed for isotopically labelled cytochrome c peroxidase 30 and Mb, 26 the difference in frequency between the 396 cm -1 and the 436 cm -1 bands is, to our knowledge, higher than normally observed for the splitting of the vinyl modes of other hemeproteins. 31 In the RR spectrum of the ferrous form of GsGCS 162 a band appears at 404 cm -1 . This band is assigned to a vinyl bending mode together with the band at 436 cm -1 .…”

Raman Spectroscopy of Blood and Blood Components

“…34 Later, the Spiro group chemically altered sub-unit regions of hemoglobin that they suspected to be important to the protein's quaternary structure (they removed C-terminus residues from particular amino acids) and used two different RRS excitations (229 nm and 441.6 nm) to show the alterations increased hemoglobin's oxygen affinity and affected its functionality (the alterations reduced the protein's Bohr effect and its cooperativity). 35 Raman spectroscopy analyses of isolated hemoglobin and its many variants 36 have led to deeper understanding of the metalloprotein's form and function. The oxygenation of hemoglobin has continued to be of interest, and enhancement Raman spectroscopy techniques other than RRS have been applied.…”