Nagatomo S, Nagai M, Tsuneshige A, Yonetani T, Kitagawa T
Institute for Molecular Science, Okazaki National Research Institutes, Japan.
Biochemistry. 1999 Jul 27;38(30):9659-66. doi: 10.1021/bi990567w.
Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).
人α-亚硝基β-脱氧血红蛋白A，即α(NO)β(脱氧)，被认为具有T（紧张）结构，其氧亲和力极低，且α血红素的铁-组氨酸（His87）（Fe-His）键断裂。分别利用电子顺磁共振（EPR）和在235nm激发的紫外共振拉曼（UVRR）光谱，在各种条件下研究了α(NO)-血红蛋白中α血红素的Fe-His键以及α1-β2接触处的亚基间相互作用。pH 6.7的亚硝基血红蛋白给出了R结构的UVRR光谱，但在存在肌醇六磷酸（IHP）的情况下，α血红素的Fe-His键断裂，色氨酸残基的UVRR谱带表现出半T样，而酪氨酸谱带仍保持R样。在pH 5.6且存在IHP的情况下，半结合的亚硝基血红蛋白α(NO)β(脱氧)，其酪氨酸和色氨酸均给出T样UVRR光谱，但一氧化碳与它的β血红素结合（α(NO)β(CO)）后，UVRR光谱变为半T样。一氧化氮与它的β血红素结合（亚硝基血红蛋白）后，色氨酸的UVRR光谱变为70% T型，但酪氨酸的几乎仍为R型。当在存在IHP的情况下将pH提高到8.2时，亚硝基血红蛋白的UVRR光谱与一氧化碳血红蛋白的相同。这些血红蛋白的EPR光谱表明，α(NO)血红素的Fe-His键部分断裂。另一方面，在pH 8.8且不存在IHP的情况下，α(NO)β(脱氧)的UVRR光谱显示出T样UVRR光谱，但EPR光谱表明，α血红素中40 - 50%的Fe-His键是完整的。因此，很明显α血红素的Fe-His键断裂与色氨酸-β37的T样接触之间存在定性相关性。我们注意到，在α1-β2界面处酪氨酸和色氨酸残基的行为并不同步。酪氨酸残基的行为很可能是由β血红素通过His-β92（F8）→Val-β98（FG5）→Asp-β99（G1）→Tyr-α42（C7）或Tyr-β145（HC2）的连接所控制的。
