ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
O25. Modulation of the Ligand–Field Anisotropy in a Series of Ferric Low
Spin Cytochromes c Mutants from Pseudomonas aeruginosa c–551
and Nitrosomonas europaea c–552
E. Harbitz a , G. Zoppellaro a , R. Kaur b , A.A. Ensign b , K.L. Bren b , K.K. Andersson a
a Department of molecular biosciences, University of Oslo, Box 1041, Blindern, 0316, Oslo, Norway,
c Department of Chemistry, University of Rochester, Rochester, 14627–0216, New York, United States
C-type cytochromes with histidine-methionine axial heme ligation play important roles in electron-transfer
reactions and in enzymes. In this work we study cyt c from Pseudomonas aeruginosa (Pa c-551) [1],
Nitrosomonas europaea (Ne c-552) [2] and two methane oxidizing bacteria. Point mutations were induced in a
key residue (Asn64) near the Met axial ligand that have a considerable impact on both heme ligand-field strength
and in the Met orientation and dynamics (fluxionality). Ne c-552 has a ferric low spin (S=1/2) EPR signal
characterized by large g anisotropy with gmax at 3.34. In Ne c-552, deletion of Asn64 (NeN64∆) changes the
heme ligand-field from more axial to rhombic and also hindered the Met fluxionality present in the wild-type
enzyme. In Pa c-551 (gmax at 3.20) replacement of Asn64 with valine induces a decrease in the axial-strain and
changes the Met configuration. Other mutants, resulting in modifications in the length of the axial Met-donating
loop, did not result in appreciable alterations of the original ligand field, but had an impact on Met orientation,
fluxionality and relaxation dynamics. Comparison of the electronic fingerprints of these proteins reveals a linear
relation between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or
dynamics. Thus, for these His-Met axially coordinated Fe(III) the large gmax value EPR signal does not
represent a special case as is observed for bis-Histidine coordinated iron [3, 4, 5].
References:
[1] Wen, X., Bren, K. L. (2005) Heme axial methionine fluxion in Pseudomonas aeruginosa Asn64Gln
cytochrome c-551. Inorg. Chem. 44, 8587-8593
[2] Zoppellaro G., T. Teschner, E. Harbitz, S. Karlsen, V. Schünemann, A. X. Trautwein, D.M. Arciero, A.B.
Hooper, S. Ciurli, and K. K. Andersson (2006) EPR and Mössbauer Spectroscopical Studies of two c-type
Cytochromes, exhibiting HALS EPR signals. ChemPhysChem 7, 1258 - 1267
[3] Hederstedt L. and K.K. Andersson (1986) Electron Paramagnetic Resonance Spectroscopy of Bacillus
subtilis cytochrome b-558 in Escherichia coli Membranes and in Succinate Dehydrogenase Complex from B.
subtilis Membranes. J. Bacteriol. 167, 735-739
[4] Friden H., M.R. Cheesman, L. Hederstedt, K.K. Andersson, and A.J. Thomson (1990) Low temperature EPR
and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate:quinone oxidoreductase indicate bishistidine
coordination of the heme iron. Biochem. Biophys. Acta 1041, 207-215
[5] Walker, F. A. (2004) Models of the bis-histidine-ligated electron-transferring cytochromes. Comparative
geometric and electronic structure of low-spin ferro and ferrihemes. Chem. Rev. 104, 589-615
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