Resonance Raman studies of heme structural differences in subunits of deoxy hemoglobin

Abstract

Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d 4 ), and the hybrids bearing the deuterated heme in only one type of subunit, which are [␣ d4 ␤ h4 ] 2 and [␣ h4 ␤ d4 ] 2 . Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the ␥ 7 mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.

Resonance Raman study of deoxy and ligated (O2 and CO) mesoheme IX-reconstituted myoglobin, hemoglobin and its α and β subunits

“…The studies on model compounds such as nickel(II) complexes of octaethylporphyrin (NiOEP) [15][16][17], etioporphyrin-I (NiETP-I) [18], or 1,5-dihydroxy-1,5-dimethyloctaethylbacteriochlorin (Ni(HOEBC)) [19] laid out a reliable framework for the interpretation of the low-frequency modes observed in the RR spectra of heme proteins. In addition, the 14 N/ 15 N, 1 H/ 2 H, 54 Fe/ 57 Fe, and 32 S/ 34 S isotope labeling of hemes in Mb [20,21], HbA and its isolated subunits and mixed hybrids [10,22,23], cytochrome c [24], cytochrome c peroxidase [25], and cytochrome P450 [26] allowed one to confirm the reliability of the RR band assignment. The approach of specifically isotope-substituted hemes is especially useful in the case of Hb hybrids, where a and b subunits can be monitored simultaneously using the same excitation line, as clearly demonstrated previously [22,23].…”

“…In addition, the 14 N/ 15 N, 1 H/ 2 H, 54 Fe/ 57 Fe, and 32 S/ 34 S isotope labeling of hemes in Mb [20,21], HbA and its isolated subunits and mixed hybrids [10,22,23], cytochrome c [24], cytochrome c peroxidase [25], and cytochrome P450 [26] allowed one to confirm the reliability of the RR band assignment. The approach of specifically isotope-substituted hemes is especially useful in the case of Hb hybrids, where a and b subunits can be monitored simultaneously using the same excitation line, as clearly demonstrated previously [22,23]. However, it has to be emphasized that the definitive assignment of the RR bands of most natural heme proteins or those substituted with chemically modified hemes has not been available yet.…”

“…Horse-heart myoglobin (Mb) from the Sigma-Aldrich Chemical Company (Pozna n, Poland) and human HbA from a local blood centre were isolated from red blood cells according to a well-established procedure [22]. ApoMb and apoHb were prepared by the acidacetone method [37].…”

“…ApoMb and apoHb were prepared by the acidacetone method [37]. Reconstitution with mesoheme IX and mesoheme-d 12 IX was accomplished as described previously [22], by adding dropwise a 1.2 molar excess of mesoheme IX dissolved in minimum volume of 0.1 M KOH into a stirred globin solution at 0°C, while maintaining pH at 6.0. Then, the iron(III) sample was reduced with sodium dithionite in CO/Ar 2 atmosphere.…”

“…Recent studies also show that despite slight differences in the RR spectra between the a and b subunits, which arise from small structural differences, the spectra of Hb tetramers can be treated as a ''sum'' of the RR spectra of both subunits [21,22,[27][28][29], with the exclusion of the m(Fe-His) mode of deoxyHb, since the isolated subunits have properties close to the R state, while deoxyHb exists in the T state [30,31]. Surprisingly, a different behaviour, in which the RR spectra of deoxy a and b subunits of mesoHb differ markedly from the RR spectrum of deoxy mesoHb tetramer, has been reported in the literature [32].…”