The chemistry of NO is important in cellular processes of higher organisms, as well as in environmental processes. NO, NO2 AND NO3 have recently been shown to be a product of macrophages and neutrophils with cytotoxic or cytostatic effects. In environmental processes, denitrifying bacteria are important agents in managing waste, as well as being a source of nitric oxide (NO) in the atmosphere. The cellular system is largely oxidative, while in the bacterial pathway, nitrates are reductively converted eventually to N2 via NO and N2O. Copper-containing proteins from denitrifying bacteria provide an excellent system to study the chemistry of NxOy compounds and to explore factors affecting redox potentials, electron transfer pathways, and specificity. In the denitrifying bacteria Alcaligenes faecalis, a small blue copper protein (pseudoazurin, a cupredoxin) donates electrons to a copper containing nitrite reductase which produces NO if NO2 is the substrate, and peroxide if O2 is the substrate. The organism Achromobacter cycloclastes contains a similar pair of molecules in its denitrification pathway, and in addition has a copper-containing N2O reductase. We have determined the X-ray structures of pseudoaxurin from A. faecalis and nitrite reductase (NIR) from A. cycloclastes, have crystallized alternate members of each pair, and now have access to site-directed mutants of pseudoazurin and NIR from A. faecalis. We will include the X- ray structure determination of site-directed mutants of A. faecalis pseudoazurin, and Achromobacter cycloclastes NIR, and start on three new proteins: A. faecalis NIR, the multicopper A. cycloclastes N2O reductase and the hexaheme-containing nitrite reductase from D. desulfuricans. In order to gain insight into the factors affecting redox potentials we will perform calculations of electrostatic contributions to the observed increase of 139 mV in the redox potential of the pseudoazurin mutant P80A over the wild type protein, using our known structures of the oxidized and reduced forms of each. We will initiate NMR studies of the native protein in order to investigate crystallographically detected structural differences in solution, as well as to measure electron transfer self- exchange rates for comparison with other cupredoxins. Knowledge of the structures of proteins which process NxOy compounds will contribute to understanding the chemistry of important life processes, as well as fundamental processes of electron transfer and protein-protein interaction.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Washington
Anatomy/Cell Biology
Schools of Medicine
United States
Zip Code
Libeu, C A; Kukimoto, M; Nishiyama, M et al. (1997) Site-directed mutants of pseudoazurin: explanation of increased redox potentials from X-ray structures and from calculation of redox potential differences. Biochemistry 36:13160-79
Murphy, M E; Turley, S; Adman, E T (1997) Structure of nitrite bound to copper-containing nitrite reductase from Alcaligenes faecalis. Mechanistic implications. J Biol Chem 272:28455-60
Murphy, M E; Lindley, P F; Adman, E T (1997) Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions. Protein Sci 6:761-70
Kukimoto, M; Nishiyama, M; Tanokura, M et al. (1996) Studies on protein-protein interaction between copper-containing nitrite reductase and pseudoazurin from Alcaligenes faecalis S-6. J Biol Chem 271:13680-3
Murphy, M E; Turley, S; Kukimoto, M et al. (1995) Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc. Biochemistry 34:12107-17
Adman, E T; Godden, J W; Turley, S (1995) The structure of copper-nitrite reductase from Achromobacter cycloclastes at five pH values, with NO2- bound and with type II copper depleted. J Biol Chem 270:27458-74
Kukimoto, M; Nishiyama, M; Ohnuki, T et al. (1995) Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6. Protein Eng 8:153-8
Kukimoto, M; Nishiyama, M; Murphy, M E et al. (1994) X-ray structure and site-directed mutagenesis of a nitrite reductase from Alcaligenes faecalis S-6: roles of two copper atoms in nitrite reduction. Biochemistry 33:5246-52
Adman, E T; Mather, M W; Fee, J A (1993) Molecular modeling studies on the proposed NaCl-induced dimerization of Chromatium vinosum high-potential iron protein. Biochim Biophys Acta 1142:93-8
Grossmann, J G; Abraham, Z H; Adman, E T et al. (1993) X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution. Biochemistry 32:7360-6

Showing the most recent 10 out of 21 publications