The hydrogen-bonding network in heme oxygenase also functions as a modulator of enzyme dynamics: chaotic motions upon disrupting the H-bond network in heme oxygenase from Pseudomonas aeruginosa.
Relaxation compensated Carr-Purcell-Meiboom-Gill (rc-CPMG) NMR experiments have been used to investigate micros-ms motions in heme oxygenase from Pseudomonas aeruginosa (pa-HO) in its ferric state, inhibited by CN- (pa-HO-CN) and N3- (pa-HO-N3), and in its ferrous state, inhibited by CO (pa-HO-CO). Comparative analysis of the data from the three forms indicates that the nature of the coordinated distal ligand affects the micros-ms conformational freedom of the polypeptide in regions of the enzyme far removed from the heme iron and distal ligand. Interpretation of the dynamical information in the context of the crystal structure of resting state pa-HO shows that residues involved in the network of structural hydrogen-bonded waters characteristic of HOs undergo micros-ms motions in pa-HO-CN, which was studied as a model of the highly paramagnetic S = 5/2 resting state form. In comparison, similar motions are suppressed in the pa-HO-CO and pa-HO-N3 complexes, which were studied as mimics of the obligatory oxyferrous and ferric hydroperoxide intermediates, respectively, in the catalytic cycle of heme degradation. These findings suggest that in addition to proton delivery to the nascent Fe(III)-OO(-) intermediate during catalysis, the hydrogen-bonding network serves two additional roles: (i) propagate the electronic state (reactive state) in each of the distinct steps of the catalytic cycle to key but remote sections of the polypeptide via small rearrangements in the network of hydrogen bonds and (ii) modulate the conformational freedom of the enzyme, thus allowing it to adapt to the demanding changes in axial coordination state and substrate transformations that take place during the catalytic cycle. This idea was probed by disrupting the hydrogen-bonding network in pa-HO by replacing R80 with L. NMR spectroscopic studies conducted with R80L-pa-HO-N3 and R80L-pa-HO-CO revealed that the mutant exhibits nearly global conformational disorder, which is absent in the equivalent complexes of the wild type enzyme. The "chaotic" disorder in the R80L mutant is likely related to its significantly lower efficiency to hydroxylate heme in the presence of H2O2, relative to the wild type enzyme.
Rodríguez
Juan Carlos
JC
Ralph N. Adams Institute for Bioanalytical Chemistry and Department of Chemistry, University of Kansas, Multidisciplinary Research Building, 2030 Becker Drive, Room 220 E, Lawrence, Kansas 66047, USA.
Zeng
Yuhong
Y
Wilks
Angela
A
Rivera
Mario
M
D001483:Base Sequence; D001664:Biliverdine / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D006900:Hydroxylation; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013057:Spectrum Analysis
D008024:Ligands; C099547:verdoheme; D006418:Heme; D006861:Hydrogen Peroxide; D006419:Heme Oxygenase (Decyclizing); D001664:Biliverdine