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

Eurobic9, 2-6 September, 2008, Wrocław, Poland
O3. A Single Mutation in Nitrophorins from Blood-sucking Insects
Flips the Heme Orientation by 180° around the C meso-α –C meso-α Axis
M. Knipp a , F. Yang b , R.E. Berry b , H. Zhang b , M. Vašák, c F.A. Walker b
a
Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D-45470, Mülheim an der Ruhr, Germany
e-mail: mknipp@mpi-muelheim.mpg.de
b
Department of Chemistry, University of Arizona, 1306 East University Boulevard, 85721-0041, Tucson, AZ,
USA
c
Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Heme b is the most common of the heme cofactors that are found in heme proteins with various functions.
Although the core structure (the tetrapyrrole ring) is highly symmetric, the distribution of the eight substituents
(methyl, propionate, and vinyl groups) around the aromatic macrocycle results in a significantly lower
symmetry. Insertion into the asymmetric pocket of a protein produces two different isomers A and B (see
Figure). For most heme b proteins a preference for one orientation exists, but the reasons are not clear [1].
Using 1 H NMR spectroscopy, stopped-flow kinetics, and other techniques, we found that two members of the
class of NO transporting ferriheme proteins termed nitrophorins (NPs) exhibit opposite cofactor orientation, i.e.,
NP2 stabilizes B orientation [2] whereas NP7 prefers A orientation [3]. This is also dramatically shown by CD
spectroscopy. Examination of the structures of both proteins (61% amino acid sequence identity) identified E27
in NP7, which is represented by V24 (V25) in NP2/3 (NP1/4), as a candidate to dictate the heme orientation.
Appropriate mutant proteins were generated, NP2(V24E), NP7(E27Q), and NP7(E27V), and characterized. As a
result, the heme orientation in NP2 was completely switched to A upon V24→E mutation. In good agreement,
NP7(E27V) showed an A:B ratio of ~1:3. Overall, we identified a single amino acid residue to be responsible for
the orientation of the heme b cofactor in the nitrophorins.
References:
[1] La Mar, G. N., Satterlee, J. D., De Ropp, J. S., Nuclear Magnetic Resonance of Hemoproteins; In The
Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Ed.; Academic Press, San Diego (USA), 2000;
Vol. 5, pp 185-298.
[2] Berry, R. E., Shokhireva, T. Kh., Filippov, I., Shokhirev, M. N., Zhang, H., Walker, F. A. Biochemistry 2007,
46, 6830-6843.
[3] Knipp, M., Yang, F., Berry, R. E., Zhang, H., Shokhirev, M. N., Walker, F. A., Biochemistry 2007, 46,
13254-13268.
_____________________________________________________________________
90