Resonance Raman optical activity of the imidazole–Myoglobin complex: Titrating enhancement

Abstract

AbstractResonance Raman optical activity (rROA) is a very powerful chiroptical technique for the investigation of bioactive protein cofactors in native conformation, due to its sensitivity towards any tiny conformational changes occurring at the chromophore. Twelve years ago, the rROA spectrum of myoglobin was published for the first time, revealing the strong potential of the technique for the selective study of the haem moiety. In this contribution, the use of rROA has been extended to the ligand binding properties of myoglobin via a combined approach of resonance Raman/ROA and ultraviolet‐visible absorption/electronic circular dichroism. The treatment of myoglobin with molar excess of imidazole in aqueous solution has led to the formation of a brilliant red pigment, known as imidazolylmet‐myoglobin, and revealed an interesting evolution of the visible Q bands, with the appearance of Qα at 534 nm. The use of the laser excitation of 532 nm, in resonance with the S0–S1 electronic transition (Q bands) of the sample, induced the selective Raman and ROA enhancement of certain haem vibrational modes, in a concentration‐dependent manner. Here, we highlight the sensitivity of rROA spectroscopy towards conformational changes of the haem chromophore upon interaction with the exogenous molecule, allowing us to distinguish between the bound and unligated form of the globin, a fundamental step towards the full understanding of the haem‐ligand binding process.