Département de biochimie, de microbiologie et de bio-informatique, Université Laval and PROTEO, Québec, Canada.
Biochemistry. 2011 Dec 27;50(51):11121-30. doi: 10.1021/bi201059a. Epub 2011 Dec 1.
The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe²⁺-O₂ + (•)NO → trHbN-Fe³⁺-OH₂ + NO₃⁻) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (•)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (•)NO and O₂ to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.
结核分枝杆菌截断血红蛋白 N（trHbN）具有很强的一氧化氮双加氧酶（NOD）活性（trHbN-Fe²⁺-O₂ + (•)NO → trHbN-Fe³⁺-OH₂ + NO₃⁻），可保护需氧呼吸免受（•）NO 的抑制。trHbN 的高活性部分归因于存在许多短寿命的疏水性空腔，这些空腔允许气态底物（•）NO 和 O₂ 分配和扩散到活性部位。我们使用溶液 NMR 光谱和分子动力学（MD）研究了这些腔与蛋白质动力学之间的关系。两种方法的结果均表明，该蛋白质主要是刚性的，其骨干 N-H 键矢量的运动非常有限，在皮秒到纳秒时间尺度上，这表明 trHbN 中的底物扩散和分配可能受侧链运动的控制。无模型分析还揭示了许多残基（包括位于螺旋 B 和 G 中的远端血红素口袋 Tyr33（B10））存在缓慢运动（微秒-毫秒），而这些残基在 MD 模拟中并未观察到。目前所有已知的带有所谓前-A N 端延伸的截断血红蛋白的晶体结构和分子动力学数据表明，一种稳定的α-螺旋构象在溶液中延伸。此外，最近的一项研究将前-A 螺旋归因于 NOD 活性的关键作用。然而，溶液 NMR 数据清楚地表明，在接近生理的条件下，这些残基不采用α-螺旋构象，而是显著无序，并且晶体结构中观察到的螺旋构象可能是由晶体接触诱导的。尽管前-A 的这种无序状态并不否认这些残基具有重要的功能作用，但我们的数据表明，在任何功能解释中，都不应假定这些残基具有螺旋构象。此外，未来的分子动力学模拟不应为这些残基使用初始α-螺旋构象，以避免基于 N 端残基错误初始结构的偏差。这项工作是通过溶液异核弛豫 NMR 光谱对截断血红蛋白动力学进行的首次研究。
