Magnetic resonance studies on the mitochondrial divalent cation carrier

Area of Science:

• Biochemistry
• Biophysics
• Membrane Transport

Background:

• Water proton spin relaxation enhancements (epsilon) can differentiate high-affinity metal ion binding from low-affinity binding to biological membranes.
• Manganese (Mn-2+) and Gadolinium (Gd-3+) ions are used as probes for studying cation binding and transport in biological systems.

Purpose of the Study:

• To investigate the utility of spin relaxation enhancements for discriminating high-affinity metal ion binding to biological membranes.
• To correlate Mn-2+ uptake with its binding site on the mitochondrial divalent cation pump.
• To characterize the binding and transport kinetics of Mn-2+ and Gd-3+ in mitochondria and other biological systems.

Main Methods:

• Measurement of water proton spin relaxation enhancements (epsilon) upon addition of Mn-2+ or Gd-3+ to biological samples.
• Monitoring the decay of epsilon during energy-driven ion uptake by mitochondria.
• Correlation of epsilon values with the initial velocity of Mn-2+ uptake at various temperatures and protein concentrations.
• NMR frequency-dependent measurements to determine correlation times (tau b) for bound metal ions.

Main Results:

• High-affinity Mn-2+ binding to rat liver mitochondria inner membrane yields epsilon values of approximately 11, distinguishing it from low-affinity binding.
• Energy-driven Mn-2+ uptake by mitochondria leads to a decay in epsilon, linearly correlating with uptake velocity across temperatures.
• Gd-3+ binding to mitochondria results in lower epsilon values (5-6) and slower uptake kinetics compared to Mn-2+.
• Uncouplers like carbonylcyanide-p-trifluoromethoxyphenylhydrazone decrease epsilon and uncouple energy-driven uptake, while de-energization alone does not affect epsilon significantly.
• Correlation time measurements suggest Mn-2+ binds to a mobile part of the mitochondrial carrier complex.

Conclusions:

• Spin relaxation enhancement is a reliable technique for identifying high-affinity metal ion binding to biological membranes, specifically the mitochondrial divalent cation pump.
• The observed linear correlation between epsilon and Mn-2+ uptake velocity provides a quantitative measure of cation transport.
• Metal ion binding sites and transport mechanisms in mitochondria can be elucidated using this NMR-based approach.
• The mobile nature of the metal ion binding site on the carrier complex is suggested by kinetic data.