Structural Dynamics of Myoglobin:  FTIR-TDS Study of NO Migration and Binding^†

Abstract

By using Fourier transform infrared photolysis difference spectroscopy combined with temperature derivative spectroscopy at cryogenic temperatures, we have measured infrared spectra of the stretching absorption on nitric oxide (NO) in the heme-bound and photodissociated states of ferrous and ferric nitrosyl myoglobin (MbNO) and a few site-specific Mb mutants. The NO absorption was utilized as a sensitive local probe of ligand interactions with active-site residues and movements within the protein. By comparison with results obtained in previous spectroscopic and structural studies of carbonmonoxy myoglobin (MbCO), the MbNO data were interpreted in structural terms. In the NO-bound state, conformational heterogeneity was inferred from the appearance of multiple bands, arising from different electrostatic interactions with active site residues, most importantly, His-64. In ferrous MbNO, a primary photoproduct site similar to site B of MbCO was found, as indicated by a characteristic NO stretching spectrum. In ferric MbNO, the His-64 side chain appears to interfere with trapping of NO in this site; only a very weak photoproduct spectrum was observed in Mb variants in which His-64 was present. Upon extended illumination, the photoproduct spectrum changed in a characteristic way, indicating that NO readily migrates to a secondary docking site C, the Xe4 cavity, in which the ligand performs librational motions on the picosecond time scale. This docking site may play a role in the physiological NO scavenging reaction. Surprisingly, NO cannot be trapped at all in secondary docking site D, the Xe1 cavity.

Unmasking the Janus face of myoglobin in health and disease

“…• as described by Nienhaus and co-workers in detail (Nienhaus et al, 2008). They observed conformational heterogeneity of myoglobin in NO-bound states, the migration of NO…”

Ligand diffusion in globins: simulations versus experiment

“…time resolved crystallography [23–25] and time resolved vibrational spectroscopy [26, 27]). Significant credit to the broader and renewed interest in myoglobin kinetics is due to the spectacular snapshots in time of ligand migration between internal cavities such as the DP, Xe4 and Xe1 [5, 23–25, 28–32].…”

Photoexcitation dynamics of nitric oxide bound ferric myoglobin probed by femtosecond IR spectroscopy

“…These bands, denoted to A 0 (the minor band) and A 1 (the major band), [6,7] have been suggested to arise from the presence of discrete bound-state conformations. It was proposed that the band A 1 results from an interaction between the bound NO and the lone-pair electron on a deprotonated His-64-N and the band A 0 from conformation where a His-64 side chain does not interact with NO [7,8]. At cryogenic temperature A 1 band was found to be at 1927 cm -1 for both sperm whale and horse Mbs.…”