The SIR provided d-[""""""""N]Aminolevulinic Acid.HC I; 7.07g L-[ring_2 HdTyrosine; 0.5g D,L-[ring- N2]Histidine; 0.5g The purpose of this project is to utilize FTIR difference spectroscopy to identify specific amino acids that have important roles in the mechanism of proton pumping of the heme-copper respiratory oxidases. This is a large family of enzymes, including cytochrome oxidase, that utilize the free energy made available from the reduction of dioxygen to water to make a voltage and pH gradients across the biological membrane. Different states of the enzyme that are important intermediates in the catalytic cycle can be generated in the laboratory, and FTIR difference spectroscopy can detect differences between these states. The technique is very sensitive, so that the protonation or deprotonation of a single amino acid in the protein can be detected as a change in the FTIR difference spectrum. Identifying the amino acids in the protein that are responsible for observed changes in the FTIR spectrum requires the use of stable isotopes incorporated in the enzyme. The change in mass results in interpretable shifts in the vibrational absorption bands monitored by FTIR. The labeled histidine and tyrosine have been incorporated into a bacterial heme-copper oxidase, cytochrome bo3 from E. coli. Currently, we are examining the purified enzymes containing the isotopically labeled amino acids using FTIR spectroscopy in collaboration with the group at the Integrated Spectroscopy Laboratory at LANL.
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