Characterization of the spontaneous "aging" of the heme oxygenase from the pathological bacterium Neisseria meningitidis via cleavage of the C-terminus in contact with the substrate. Implications for functional studies and the crystal structure.
Solution 1H NMR spectroscopy and mass spectrometry are utilized to characterize the irreversible "aging" of native heme oxygenase from N. meningitidis, NmHO. 2D NMR characterization of the cyanide-inhibited substrate complex shows that the C-terminal interaction between Arg208His209 and the exposed pyrrole of the protohemin substrate in the "native" NmHO complex is lost in the "aging". Mass spectrometry and N-terminal sequencing of wild type and "aged" NmHO reveal that the "aging" process involves cleavage of the Arg208His209 dipeptide. The construction of the double deletion mutant without Arg208His209 and its NMR comparison as both the resting state substrate complex and its cyanide-inhibited complex with the "aged" NmHO reveal that cleavage of the C-terminal dipeptide is the only modification during the aging. Comparison of cyanide ligand binding constants reveal a factor approximately 1.7 greater CN- affinity in the native than "aged" NmHO. The rate of protohemin degradation and its stereoselectivity are unaffected by the C-terminal truncation. However, the free alpha-biliverdin yield in the presence of desferrioxamine is significantly increased in the "aged" NmHO and its deletion mutant relative to WT, arguing for a role of the NmHO C-terminus in modulating product release. The facile cleavage of Arg208His209 in the resting state complex, with a half-life of approximately 24 h at 25 degrees C, suggests that previous characterization of NmHO may have been carried out on a mixture of native and "aged" NmHO, and may account for the "lost" C-terminal residues in the crystal structures.
Department of Chemistry, University of California, Davis, California 95616, USA.
Yangzhong
Y
Liu
Li-Hua
LH
Ma
Xuhong
X
Zhang
Tadashi
T
Yoshida
James D
JD
Satterlee
Gerd N
GN
La Mar
D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009345:Neisseria meningitidis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D017931:DNA Primers; D006419:Heme Oxygenase (Decyclizing)