Heme oxygenases from the bacterial pathogens
Neisseriae meningitidis (
nm-HO) and
Pseudomonas aeruginosa (
pa-HO) share significant sequence identity (37%). In
nm-HO, biliverdin IX
is the sole product of the reaction, whereas
pa-HO yields predominantly biliverdin IX
. We have previouslyshown by NMR that the in-plane conformation of the heme in
pa-HO is significantly different from thatof
nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan,G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman,M. A., and Rivera, M. (2002)
J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, wehave extended these studies to investigate the role of the distal helix by preparing a chimera of
nm-HO(
nm-HOch), in which distal helix residues 107-142 of
nm-HO have been replaced with the correspondingresidues of the
-regioselective
pa-HO (112-147). Electronic absorption spectra, resonance Raman andFTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximalHis ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalyticturnover of the
nm-HOch-heme complex yields almost exclusively
-biliverdin and a small but reproducibleamount of
-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimericprotein likely stems from a dynamic equilibrium between two alternate in-plane conformations of theheme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric proteinin an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformerconducive to
-meso hydroxylation.