Angewandte
Chemie
once the uncoupling pathways are suppressed by localizing
ethane close to the ferryl oxygen atom.
Received: November 16, 2004
Revised: March 17, 2005
Published online: May 24, 2005
Keywords: alkane oxidation · heme proteins · iron ·
.
metalloenzymes · protein engineering
Figure 3. GC analysis of ethane oxidation assays with the EB/L294M/
T185M/L1358P/G248A mutant. The controls with no ethane or no
NADH added show the residual background peak from ethanol
(<1 mm) present in the water used to prepare the buffers. The appar-
ent difference in the ethanol product peak areas for two separate incu-
bations was corrected by the ratio to the pentan-1-ol internal standard
peak. tret =retention time.
[1] J. D. Lipscomb, Annu. Rev. Microbiol. 1994, 48, 371.
[2] J. C. Murrell, B. Gilbert, I. R. McDonald, Arch. Microbiol. 2000,
173, 325.
[3] D. A. Kopp, S. J. Lippard, Curr. Opin. Chem. Biol. 2002, 6, 568.
[4] J. G. Leahy, P. J. Batchelor, S. M. Morcomb, FEMS Microbiol.
Rev. 2003, 27, 449.
[5] S. I. Chan, K. H. Chen, S. S. Yu, C. L. Chen, S. S. Kuo,
Biochemistry 2004, 43, 4421.
[6] Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd
ed. (Ed.: P. R. Ortiz de Montellano), Plenum, New York, 1995.
[7] M. J. Cryle, J. E. Stok, J. J. De Voss, Aust. J. Chem. 2003, 56, 749.
[8] I. C. Gunsalus, G. C. Wagner, Methods Enzymol. 1978, 52, 166.
[9] S. G. Bell, J.-A. Stevenson, H. D. Boyd, S. Campbell, A. D.
Riddle, E. L. Orton, L.-L. Wong, Chem. Commun. 2002, 490.
[10] S. G. Bell, E. Orton, H. Boyd, J.-A. Stevenson, A. Riddle, S.
Campbell, L.-L. Wong, Dalton Trans. 2003, 2133.
[11] P. J. Loida, S. G. Sligar, Protein Eng. 1993, 6, 207.
[12] P. J. Loida, S. G. Sligar, Biochemistry 1993, 32, 11530.
[13] M. W. Peters, P. Meinhold, A. Glieder, F. H. Arnold, J. Am.
Chem. Soc. 2003, 125, 13442.
[14] We had reported earlier that one molecule in the P1 asymmetric
unit in the X-ray crystal structure of the F87W/Y96F/V247L
mutant complexed with 1,3,5-trichlorobenzene had low sub-
strate occupancy, and that the density above the heme could be
modeled with three water molecules, but their locations could
not be refined (X. Chen, A. Christopher, J. P. Jones, S. G. Bell, Q.
Guo, F. Xu, Z. Rao, L. L. Wong, J. Biol. Chem. 2002, 277, 37519).
The structure of the substrate-free form of the mutant was
therefore determined. Procedures for crystallization of the
mutant at 291 K by the hanging drop vapor diffusion method
and data collection and refinement were as reported previously.
Crystals of the F87W/Y96F/V247L mutant belonged to the space
group P21, with unit-cell dimensions: a = 66.8 ꢀ, b = 62.1 ꢀ, c =
94.9 ꢀ, a = 908, b = 90.58, g = 908. A total of 236028 reflections
were measured, with Rmerge of 7.6% for 45978 unique reflections
and 99.9% completeness (50–2.1 ꢀ). Data were collected to
100% completeness in the highest resolution shell. The structure
was solved by molecular replacement based on the crystal
structure of wild-type P450cam (PDB code: 2CPP), but with the
camphor removed. After initial refinement, the difference
Fourier map for both molecules in the unit cell showed well-
defined triangle-shaped electron density above the heme group
that was modeled by three water molecules. The final refinement
parameters were Rwork = 19.1% and Rfree = 24.3%. Full details
will be published elsewhere.
were low spin. The six-water cluster in substrate-free P450cam
has strong hydrogen bonding, and the heme-bound water
ligand is likely to have significant hydroxide ion character.
Raag and Poulos noted the importance of hydrogen bonding
to the heme ligand water: wild-type P450cam with non-natural
substrates bound could have a six-coordinate heme, yet the
iron center was high spin. Therefore, the presence of this sixth
ligand on its own was not sufficient to bring the heme to a low-
spin state.[22] Model compound studies have also shown that
the P450 heme group is low spin if the axial water ligand is
hydrogen bonded to other groups, but is high spin if there is
no such hydrogen bonding.[23] The axial water ligand in the
F87W/Y96F/V247L mutant is hydrogen bonded to two other
waters, and the heme is low spin. As the EB/L294M/T185M/
L1358P/G248A mutant contains five additional bulky sub-
stitutions within the active site, it is very likely that the three-
water cluster observed in the F87W/Y96F/V247L mutant
would be perturbed significantly, leaving only two or even one
water molecule in the active site. Fewer active site water
molecules and weakened hydrogen bonding allows ethane to
bind more readily, and in this case, to induce a heme-group
shift to > 90% high spin and a fast NADH turnover rate.
Hence the EB/L294M/T185M/L1358P/G248A mutant has not
only the interesting property of ethane oxidation, but its
active site water structure could provide new insight into the
origin of the heme spin state equilibrium in P450 enzymes.
In summary, we have engineered a cytochrome P450
enzyme to oxidize ethane to ethanol by decreasing the active
site volume with bulky substitutions, and by altering the
hydrogen bonding to the proximal ligand by the L358P
mutation first reported by Morishima.[16,17] The high spin
heme content of the EB/L294M/T185M/L1358P/G248A
mutant in the absence of substrate suggests that it may also
be a useful platform for structure–function studies of P450
enzymes. Finally, the high NADH oxidation rate of this
mutant with ethane suggests that, as we had shown for n-
butane and propane, fast ethane oxidation will be possible
[15] T. L. Poulos, B. C. Finzel, A. J. Howard, Biochemistry 1986, 25,
5314.
[16] S. Yoshioka, S. Takahashi, K. Ishimori, I. Morishima, J. Inorg.
Biochem. 2000, 81, 141.
[17] S. Nagano, T. Tosha, K. Ishimori, I. Morishima, T. L. Poulos, J.
Biol. Chem. 2004, 279, 42844.
[18] T. L. Poulos, B. C. Finzel, A. J. Howard, J. Mol. Biol. 1987, 195,
687.
Angew. Chem. Int. Ed. 2005, 44, 4029 –4032
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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