J. Biol. Chem., Vol. 266, Issue 4, 2210-2218, 02, 1991
Extended x-ray absorption fine structure and electron paramagnetic resonance of nitrous oxide reductase from Pseudomonas aeruginosa strain P2
CK SooHoo, TC Hollocher, AF Kolodziej, WH Orme-Johnson and G Bunker
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
The copper centers of nitrous oxide reductase from Pseudomonas aeruginosa
strain P2 were studied by x-ray and electron paramagnetic resonance (EPR)
spectroscopy. The enzyme is dimeric and contains four Cu atoms and about
seven cysteine residues/subunit of Mr = 73,000. The extended x-ray
absorption fine structure (EX-AFS) spectrum was analyzed for enzyme as
isolated (oxidized or slightly reduced), enzyme exposed briefly to air,
reduced enzyme, and enzyme at pH 7 after having been activated by standing
at pH 10. The average Cu ligand environment in the first shell was best
modeled for all forms of the enzyme by a combination of N/O and S atoms at
a total coordination number between 3 and 4 and bond distances ranging from
1.96-2.03 A for Cu-N/O and 2.20- 2.25 A for Cu-S. The data could be fit
without using Cu-Cu interactions. Overall the results are similar to those
reported for the enzyme for Pseudomonas stutzeri (Scott, R. A., Zumft,
W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A.
86, 4082- 4086). The first derivative EPR spectra of the Cu(II) centers at
15 and 45 K were qualitatively similar among enzyme as isolated and enzyme
exposed to N2O or air. These three nominally oxidized samples showed an
axial signal with g perpendicular = 2.03 and g parallel = 2.15-2.16.
Hyperfine structure was observed in both the g parallel and g perpendicular
regions with splittings of 43 and 25 gauss, respectively. These hyperfine
components are attributed to exchange coupled Cu(I)- Cu(II) S = 1/2
(half-met) centers. In the enzyme as isolated and after exposure to N2O,
about 3/4 of the Cu was EPR silent, whereas after exposure to air the
signal integrated to about half the Cu concentration. The EPR spectrum of
enzyme activated at pH 10 but frozen at pH 7 was a composite of spectra
from activated and inactive species. The activated species presented a
complex set of narrow hyperfine components which may arise from
contributions from more than one species of half-met center.
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