(An experiment in time-resolved step-scan FT-IR for use in dynamic photophysical studies of cytochrome-C oxidase and other heme proteins). Final report

摘要

Development of a time-resolved step-scan FT-IR for use in dynamic photophysical studies of cytochrome-C oxidase and other heme proteins is described. An instrument was assembled which incorporated a step-scan FT-IR spectrometer developed at Duke and several components on loan from LANL, including particularly a frequency doubled Nd-YAG pump laser and a pair of gated integrators. The specific objective was to obtain information on the slower steps ((mu)s-ms) of the reassociation of CO with the heme iron of cytochrome-C oxidase and myoglobin after its photodissociation by a 10 ns pulse from the ND-YAG laser and to correlate the CO dynamics with associated conformational dynamics in the protein, as indicated by changes in the CO and amide vibrational bands. The step-scan FT-IR probe allows time-resolved spectra to be obtained over the entire mid-IR region. Although the D(sub 2)O solvent occludes much of this region, observation in the CO stretching and Amide I, II, and III mode regions is still possible in the D(sub 2)O windows. Initial results were obtained on cytochrome-C oxidase, but, because of the sample requirements, the more readily available carbonmonoxymyoglobin (MbCO) was chosen for study during the rest of the technique development. The initial results on myoglobin are encouraging as to the original objective of simultaneously monitoring both the reassociation of the CO and the protein conformational changes, but comparison of the FT-IR results with those obtained from the same sample at LANL by use of a tunable IR diode laser show significant differences. There is strong indication that the total laser power required to get a useful dynamic difference signal in the FT-IR experiment is causing a significant temperature-induced reversible conformational change in the protein which is much larger than that associated with the dissociation/reassociation of the CO.