Dr. Wayne D. Frasch

My laboratory is investigating the structure and catalytic mechanism of enzymes that are crucial to the process of photosynthetic energy conversion. The energy-transducing complex, or coupling factor, is
required by virtually every living organism to catalyze the conversion of energy from electrochemical potential into ATP, a form of energy that is used to drive most cellular process. We are using site-directed mutagenesis in Chlamydomonas to introduce mutations of single amino acid residues of couplingfactor to assess their relative importance in the catalytic mechanism. This enzyme requires divalent metal ions as cofactors. We are identifying the amino acids to which these metals bind using the metal vanadyl as a spectroscopic probe. The EPR signal that arises from the vanadyl changes in predictable ways when the groups that bind the metal change. Through a combination of the molecular genetics and the spectroscopy of the bound vanadyl, we are able to identify the specific amino acids that are directly involved with metal-binding. We are also following the changes in the binding of the metals that occur as the enzyme steps through its catalytic mechanism. These experiments are providing new insights into the means by which the energy obtained from the hydrolysis of ATP can be converted into the physical action of pumping a proton in a unilateral direction. Determining the mechanism of this micro-pump also has direct applications to the mechanisms of other important life processes where the chemical energy released from ATP hydrolysis is converted into mechanical force. Some of these processes include the mechanism of actino-myosin in the process of muscle contraction as well as the function of G-proteins, which mediate the communication of cellular responses to initiate gene expression.

A second area of study concerns the oxygen-evolving complex in photosynthesis which is responsible for most of the oxygen in the atmosphere. We recently developed a procedure whereby the calcium
ions required for this process can be substituted with other metals that can be used as probes of the structure and mechanism. Using these metal-substituted preparations we have found that at least one of the calcium ions is located a few Angstroms from the manganese ions that are responsible for oxidizing water to molecular oxygen. These preparations are providing new insights into the relationship between the structure of this metal cluster and its ability to catalyze this important reaction.

The F1-ATPase is a molecular motor. We are currently attaching this enzyme to chips and are examining rotation of the gamma subunit that is driven by the ATPase reaction. The nanoscale fluidity of the surrounding solution is being investigated.