Pressure induced structural fluctuations in hemoglobin, studied by EPR-spectroscopy

Area of Science:

• Biophysics
• Spectroscopy
• Materials Science

Background:

• Electron Paramagnetic Resonance (EPR) is a powerful technique for studying molecules with unpaired electrons.
• Understanding protein dynamics under pressure is crucial for biological function.
• Previous EPR studies were limited in their ability to probe liquid samples at high pressures.

Purpose of the Study:

• To construct and validate a novel quartz cavity for pressure-dependent EPR measurements on liquid samples.
• To investigate the pressure-induced changes in the mobility of spin labels attached to hemoglobin.
• To correlate these mobility changes with the electronic spin state of the iron-porphyrin complex in hemoglobin.

Main Methods:

• Construction of a quartz-based cavity capable of withstanding pressures up to 0.6 GPa.
• EPR measurements on spin-labeled horse hemoglobin derivatives in both ferric and ferrous states.
• Analysis of the second derivative EPR spectra to assess label mobility and spin state.

Main Results:

• The newly developed quartz cavity successfully enabled EPR measurements on liquid samples up to 0.6 GPa.
• Changes in the EPR spectra indicated alterations in the mobility of the spin label attached to hemoglobin.
• These mobility changes were found to be independent of the oxidation state (ferric/ferrous) of the iron-porphyrin complex.

Conclusions:

• The study demonstrates the utility of the pressure-dependent EPR setup for probing molecular dynamics in liquids.
• The observed changes in spin label mobility suggest significant structural fluctuations within the globin protein matrix under pressure.
• These protein structural dynamics are decoupled from the electronic spin state transitions of the heme iron.