Resonance Raman Spectra of an O2-Binding H-NOX Domain Reveal Heme Relaxation upon Mutation

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• 作者：Rosalie Tran ; Elizabeth M. Boon ; Michael A. Marletta ; Richard A. Mathies
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：September 15, 2009
• 年：2009
• 卷：48
• 期：36
• 页码：8568-8577
• 全文大小：917K
• 年卷期：v.48,no.36(September 15, 2009)
• ISSN：1520-4995

文摘

Resonance Raman spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O₂-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., γ₁₁, γ₁₂, γ₁₃, and γ₁₅) in the 400−750 cm⁻¹ region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, ν₃, ν₂, and ν₁₀. In addition, all three mutants exhibited some degree of heme conformational heterogeneity based on several broad skeletal markers (e.g., ν₁₀) in the high frequency region. These results are comparable to those observed by Olea et al. for Tt H-NOX P115A in crystal form, where four different heme structures were determined from a single unit cell. On the basis of the resonance Raman spectra, it is clear that the actual heme conformation for Tt H-NOX P115A in solution is considerably more relaxed than that of the WT protein, with increased flexibility within the protein pocket, allowing for rapid sampling of alternate conformations.