Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, is a dimeric haem-protein whose biological role is still unknown. As other globins, protoglobin can bind O2, CO and NO reversibly in vitro, but it displays specific functional and structural properties within members of the hemoglobin superfamily. CO binding to and dissociation from the haem occurs through biphasic kinetics, which arise from binding to (and dissociation from) two distinct tertiary states in a ligation-dependent equilibrium. From the structural viewpoint, protoglobin-specific loops and a N-terminal extension of 20 residues completely bury the haem within the protein matrix. Thus, access of small ligand molecules to the haem is granted by two apolar tunnels, not common to other globins, which reach the haem distal site from locations at the B/G and B/E helix interfaces. Here, the roles played by residues Trp(60)B9, Tyr(61)B10 and Phe(93)E11 in ligand recognition and stabilization are analyzed, through crystallographic investigations on the ferric protein and on selected mutants. Specifically, protein structures are reported for protoglobin complexes with cyanide, with azide (also in the presence of Xenon), and with more bulky ligands, such as imidazole and nicotinamide. Values of the rate constant for cyanide dissociation from ferric MaPgb-cyanide complexes have been correlated to hydrogen bonds provided by Trp(60)B9 and Tyr(61)B10 that stabilize the haem-Fe(III)-bound cyanide. We show that protoglobin can strikingly reshape, in a ligand-dependent way, the haem distal site, where Phe(93)E11 acts as ligand sensor and controls accessibility to the haem through the tunnel system by modifying the conformation of Trp(60)B9.来自产甲烷八叠球菌C2A（Methanosarcina acetivorans C2A）的原珠蛋白（Protoglobin，MaPgb）是一种严格厌氧的产甲烷古菌（Archaea）来源的二聚体血红素（haem）蛋白，其生物学功能迄今仍未阐明。与其他珠蛋白类似，原珠蛋白在体外可可逆结合氧气（O₂）、一氧化碳（CO）与一氧化氮（NO），但在血红蛋白超家族（hemoglobin superfamily）成员中展现出独特的功能与结构特性。CO与血红素的结合及解离过程呈现双相动力学特征，该过程源于配体依赖平衡下两种不同三级状态之间的结合与解离事件。从结构层面分析，原珠蛋白特有的环结构以及一段含20个残基的N端延伸序列，可将血红素完全包裹于蛋白质基质内部。因此，小分子配体需通过两条非极性隧道方可抵达血红素远端结合位点——这类隧道并非其他珠蛋白所共有，其开口位于B/G螺旋接口与B/E螺旋接口处。本研究通过对三价铁蛋白及其选定突变体开展晶体学研究，分析了Trp(60)B9、Tyr(61)B10与Phe(93)E11残基在配体识别与稳定过程中所发挥的作用。具体而言，本文报道了原珠蛋白与氰化物、叠氮化物（亦可在氙气存在条件下）以及咪唑、烟酰胺等大体积配体形成的复合物的蛋白结构。三价铁MaPgb-氰化物复合物的氰化物解离速率常数，与Trp(60)B9、Tyr(61)B10所形成的、可稳定血红素Fe(III)结合氰化物的氢键作用密切相关。研究表明，原珠蛋白可通过配体依赖的方式显著重塑血红素远端位点：其中Phe(93)E11可作为配体传感器，并通过改变Trp(60)B9的构象，调控隧道系统对血红素的配体可及性。