Oxygen distribution and migration within Mbdes Fe and Hbdes Fe. Multifrequency phase and modulation fluorometry...

Area of Science:

• Biophysics
• Photochemistry
• Protein Dynamics

Background:

• Porphyrin fluorescence in proteins like myoglobin and hemoglobin is sensitive to molecular interactions.
• Oxygen is a known fluorescence quencher, but its dynamic behavior within protein interiors requires detailed investigation.

Purpose of the Study:

• To investigate the dynamic quenching of porphyrin fluorescence in iron-free myoglobin (Mbdes Fe) and hemoglobin (Hbdes Fe) by oxygen.
• To develop and apply a general model for dynamic fluorescence quenching in globular proteins.

Main Methods:

• Utilized a multifrequency cross-correlation phase fluorometer to measure fluorescence intensity and lifetime.
• Applied varying oxygen pressures to Mbdes Fe and Hbdes Fe samples.
• Investigated the effect of increased viscosity (using 40% sucrose) on quenching dynamics.

Main Results:

• Oxygen pressure induced a transition from single to double exponential fluorescence decay in Mbdes Fe and Hbdes Fe.
• A dynamic quenching model successfully described the acquisition (k+), exit (k-), and migration (chi) rates of oxygen within the proteins.
• These rates (k+, k-, chi) were distinct for Mbdes Fe and Hbdes Fe and were influenced by changes in viscosity.

Conclusions:

• The study provides a quantitative model for understanding oxygen quenching in globular proteins.
• The distinct quenching parameters for Mbdes Fe and Hbdes Fe highlight protein-specific dynamics.
• The findings emphasize the importance of considering quencher migration and diffusion within proteins for accurate interpretation of fluorescence quenching studies.