Palaniappan V, Bocian D F
Department of Chemistry, University of California, Riverside 92521-0403.
Biochemistry. 1994 Nov 29;33(47):14264-74. doi: 10.1021/bi00251a039.
The pH dependence of the absorption and resonance Raman (RR) spectra of the deoxy and met forms of myoglobin (Mb) has been examined in detail. The spectral data were acquired at a number of different pHs (12) in the 2.6-7.6 range. RR spectra were obtained for both the low- and high-frequency regions by using a variety of excitation wavelengths ranging from the UV to the green. The data obtained for deoxyMb indicate that a spectroscopically distinct intermediate (I') exists at equilibrium in the pH 3.5-4.5 range. The I'-form of metMb could not be identified. The Soret absorption maximum of the I'-form of deoxyMb is at approximately 426 nm compared with the value of 435 observed for the native (N) form and 383 nm observed for the so-called unfolded (U') form which occurs in the pH 2.6-3.5 range. The absorption and vibrational spectra of the I'-form of deoxyMb observed at equilibrium are very similar to those of the intermediate that appears within a few milliseconds in pH-jump experiments. The RR data indicate that the structure of the heme group in the I'-form is distinctly different from that of either N- or U'-forms. The iron-histidine bond, characteristic of the N-form, is ruptured in both the I'- and U'-forms as is evidenced by the absence of the RR band due to the stretching vibration of this unit. In the I'-form, the histidine ligand is replaced by a relatively strongly bound, exchangeable water molecule. This ligand is absent in the U'-form. The aquo ligand of the five-coordinate heme in the I'-form is identified by a RR band at 411 cm-1 which undergoes a 15-17 cm-1 downshift in deuteriated buffer solutions. In contrast, none of the RR bands of the N- and U'-forms exhibit any significant isotope sensitivity. The properties of the I'-form and the conditions under which it is generated strongly suggest that this form corresponds to the molten globule intermediate of apoMb.
已详细研究了肌红蛋白（Mb）的脱氧形式和高铁形式的吸收光谱及共振拉曼（RR）光谱的pH依赖性。光谱数据是在2.6 - 7.6范围内的多个不同pH值（12个）下采集的。通过使用从紫外到绿色的各种激发波长，获得了低频和高频区域的RR光谱。脱氧肌红蛋白（deoxyMb）获得的数据表明，在pH 3.5 - 4.5范围内，一种光谱上不同的中间体（I'）在平衡状态下存在。高铁肌红蛋白（metMb）的I'形式无法识别。脱氧肌红蛋白I'形式的Soret吸收最大值约为426 nm，而天然（N）形式为435 nm，在pH 2.6 - 3.5范围内出现的所谓解折叠（U'）形式为383 nm。在平衡状态下观察到的脱氧肌红蛋白I'形式的吸收光谱和振动光谱与在pH跃变实验中几毫秒内出现的中间体的光谱非常相似。RR数据表明，I'形式的血红素基团结构与N形式或U'形式明显不同。N形式特有的铁 - 组氨酸键在I'形式和U'形式中均断裂，这由该单元拉伸振动导致的RR带缺失所证明。在I'形式中，组氨酸配体被一个结合相对较强、可交换的水分子取代。U'形式中不存在这种配体。I'形式中五配位血红素的水合配体通过411 cm⁻¹处的RR带识别，该带在重水缓冲溶液中发生15 - 17 cm⁻¹的下移。相比之下，N形式和U'形式的RR带均未表现出任何显著的同位素敏感性。I'形式的性质及其产生的条件强烈表明，这种形式对应于脱辅基肌红蛋白的熔球中间体。
