EPR OF HEMOPROTEINS &MODELS W/ HISTIDINE TYROSINE LIGATION

  • Marcinkeviciene, Jovita (PI)
  • Friedman, Joel M. (PI)
  • Ash, David E. (PI)
  • Gerfen, Gary J. (PI)

Project: Research project

Project Details

Description

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S, 3S)-3-methylasparate. These compounds contained the isotopes at C-2, C-3 or C-4 of the carbon chain: [2-2H], [3-2H2], [4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by EPR spectroscopy and revealed a similar signal characterized by g'xy=2.1,g'z=1.985 and A'=5.0mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6+/- 0.9A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principle contributor to the radical pair to be identiied as the 4-glytamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e. the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond o the coenzyme B21.
StatusFinished
Effective start/end date10/1/959/30/99

Funding

  • National Center for Research Resources

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  • BIOTECHNOLOGY RESOURCE IN PULSED EPR SPECTROSCOPY

    Peisach, J., Aisen, P., Armstrong, W. H., Ash, D. E., Astroff, N., Avdievich, N., Baker, T. A., Banerjee, R. V., Bender, C., Benkovic, S., Brewer, C. F., Brewer, F., Britt, D., Cammack, R., Caradonna, J., Chan, J., Colaneri, M., Danzinger, N. R., Das, T., Dooley, D., Dyson, H. J., Ettinger, M., Fabry, M. E., Fee, J. A., Frank, N. H. A., Friedman, J. M., Gerfen, G. J., Goutermann, M., Guajardo, G. B., Janson, E., Kamp, N., Kozarich, J., Lee, C. C., Lippai, I., Magliozzo, R. S., Mason, R., McMillin, D., Michael, D., Mildvan, A., Mims, W. B., Narasimhulu, S., Nersissian, A., Ohnishi, T., Palmer, G., Que, L., Rousseau, D. L., Sam, J., Scholes, C., Scholes, C. P., Schramm, V. L., Shaka, O., Shanklin, J., Sinclair, P., Singel, D., Stafford, D. W., Stegeman, J., Stubbe, N. J. A., Stubbe, J., Tipton, P. A., Villafranca, N. J. J., Walter, P., Wang, W., Wittaker, J., Zweier, J. L., Belford, L., Beltramini, M., Bowman, M., Chance, M., Clarkson, R., Coffino, A., Crowder, M., Gluck, M. R., Klein, M., Klinman, J., Krzystek, Y., Licht, S., Marcinkeviciene, J., Mcdermott, A., Ming, L., Peisach, J., Ranney, H., Sands, R., Sheik, A., Stubbe, J., Sumuni, R., Tsuneshige, A., Valentine, J., Vieso, V., Wirt, M., Wong, K. & Zhang, C.

    9/30/854/30/00

    Project: Research project