School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
Department of Chemistry, Shanghai Key Lab of Chemical Biology for Protein Research & Institute of Biomedical Science, Fudan University, Shanghai 200433, China.
J Inorg Biochem. 2018 May;182:141-149. doi: 10.1016/j.jinorgbio.2018.02.011. Epub 2018 Feb 19.
The structure and function of heme proteins are regulated by diverse post-translational modifications including heme-protein cross-links, with the underlying mechanisms not well understood. In this study, we introduced a Cys (K42C) close to the heme 4-vinyl group in sperm whale myoglobin (Mb) and solved its X-ray crystal structure. Interestingly, we found that K42C Mb can partially form a Cys-heme cross-link (termed K42C Mb-X) under dithiothreitol-induced reductive conditions in presence of O, as suggested by guanidine hydrochloride-induced unfolding and heme extraction studies. Mass spectrometry (MS) studies, together with trypsin digestion studies, further indicated that a thioether bond is formed between Cys42 and the heme 4-vinyl group with an additional mass of 16 Da, likely due to hydroxylation of the α‑carbon. We then proposed a plausible mechanism for the formation of the novel Cys-heme cross-link based on MS, kinetic UV-vis and electron paramagnetic resonance (EPR) studies. Moreover, the Cys-heme cross-link was shown to fine-tune the protein reactivity toward activation of HO. This study provides valuable insights into the post-translational modification of heme proteins, and also suggests that the Cys-heme cross-link can be induced to form in vitro, making it useful for design of new heme proteins with a non-dissociable heme and improved functions.
血红素蛋白的结构和功能受多种翻译后修饰调控，包括血红素蛋白交联，但其潜在机制尚不清楚。在这项研究中，我们在抹香鲸肌红蛋白（Mb）中靠近血红素 4-乙烯基的位置引入了一个半胱氨酸（K42C），并解析了其 X 射线晶体结构。有趣的是，我们发现 K42C Mb 在存在 O 的情况下，在二硫苏糖醇诱导的还原条件下可以部分形成 Cys-血红素交联（称为 K42C Mb-X），这一点可通过盐酸胍诱导的变性和血红素提取研究得到证实。质谱（MS）研究，以及胰蛋白酶消化研究，进一步表明 Cys42 和血红素 4-乙烯基之间形成了硫醚键，分子量增加了 16 Da，可能是由于α-碳的羟化。然后，我们基于 MS、动力学 UV-vis 和电子顺磁共振（EPR）研究提出了一种形成新型 Cys-血红素交联的合理机制。此外，Cys-血红素交联可微调蛋白质对 HO 激活的反应性。该研究为血红素蛋白的翻译后修饰提供了有价值的见解，并表明 Cys-血红素交联可以在体外诱导形成，这对于设计具有不可解离血红素和改善功能的新型血红素蛋白非常有用。
