Faculty of Life Sciences, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, UK.
FEBS J. 2013 Dec;280(23):6070-82. doi: 10.1111/febs.12526. Epub 2013 Oct 11.
The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.
血红素蛋白区分双原子分子的能力，以及随后对其选择配体的亲和力，是生命存在的基础。这些过程通常由蛋白质中的精确结构排列控制，血红素口袋残基驱动反应性和特异性。细胞色素 c' 就是这样一种蛋白质，它能够在血红素的相对面上结合一氧化氮（NO）和一氧化碳（CO），这一特性与可溶性鸟苷酸环化酶共享。与可溶性鸟苷酸环化酶一样，细胞色素 c' 也完全将 O2 排除在结合口袋之外。先前的研究表明，NO 结合机制受近端精氨酸残基（R124）和远端亮氨酸残基（L16）的调节。在这里，我们使用各种跨越 UV-vis 光谱整个飞秒-秒时间范围以及飞秒-纳秒时间范围的时间分辨光谱技术，研究了这些残基在血红素结合环境中维持对 NO 亲和力的作用。我们的研究结果表明，在野生型细胞色素 c' 中，激发后紧密调节的 NO 再结合事件受到 R124A 变体的影响。在 R124A 变体中，振动和电子变化连续跨越所有时间尺度（从飞秒到秒），与野生型细胞色素 c' 和 L16A 变体形成对比。基于这些发现，我们提出了细胞色素 c' R124A 变体的 NO（再）结合机制，与野生型细胞色素 c' 不同。从更广泛的角度来看，这些发现强调了血红素口袋结构在维持血红素蛋白对其选择配体的反应性方面的重要性，并展示了跨越广泛时间范围的光谱探针的强大功能。
