The techniques of magnetic susceptibility and rapid reaction kinetics will be used to study the thermodynamic and kinetic linkages between changes in protein conformation and iron electronic configuration in hemoglobin A, other mutant hemoglobins, and various metalloproteins. To accomplish this we have developed a unique high-sensitivity time-resolved magnetic susceptometer. This instrument, which is based on recent advances in superconducting technology, is nearly 100 times more sensitive that conventional susceptometers, and provides microsecond time resolution to make possible the study of the rapid kinetics of spin state changes in hemoglobins and other metalloproteins. This new Time-Resolved Magnetic Susceptibility (TREMS) method also can be used to investigate the influence of subunit association on spin state dynamics in dimeric and tetrameric hemeproteins or in extended assemblies such as hemoglobin S. While the mechanism of cooperative oxygen binding to hemoglobin is still not known, current proposals invoke coupling of spin state changes to conformational changes. We propose some critical tests of the Perutz stereochemical model of cooperativity, and measurements to quantitate the linkage between quaternary structure and spin state. These magnetic studies will be carried out in parallel with both equilibrium and kinetic optical spectroscopic measurements of the same allosterically modifiable hemoglobins. A number of other metalloproteins will be studied to answer specific questions of electronic configuration and structure. For example, measuring the antiferromagnetic coupling in the invertebrate oxygen carriers hemocyanin and hemerythrin will elucidate the mode by which a single O2 is bound to two metal ions. The magnetic properties of other hemeproteins and iron-sulfur proteins will also be explored.
