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 aerugin
详细信息 查看全文
- 作者：Juan Carlos Rodrí ; guez ; Yuhong Zeng ; Angela Wilks ; Mario Rivera
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：September 26, 2007
- 年：2007
- 卷：129
- 期：38
- 页码：11730 - 11742
- 全文大小：965K
- 年卷期：v.129,no.38(September 26, 2007)
- ISSN：1520-5126
文摘
Relaxation compensated Carr-Purcell-Meiboom-Gill (rc-CPMG) NMR experiments have beenused to investigate s-ms motions in heme oxygenase from Pseudomonas aeruginosa (pa-HO) in itsferric 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 thecoordinated distal ligand affects the s-ms conformational freedom of the polypeptide in regions of theenzyme far removed from the heme iron and distal ligand. Interpretation of the dynamical information inthe context of the crystal structure of resting state pa-HO shows that residues involved in the network ofstructural hydrogen-bonded waters characteristic of HOs undergo s-ms motions in pa-HO-CN, whichwas studied as a model of the highly paramagnetic S = 5/2 resting state form. In comparison, similar motionsare suppressed in the pa-HO-CO and pa-HO-N3 complexes, which were studied as mimics of the obligatoryoxyferrous and ferric hydroperoxide intermediates, respectively, in the catalytic cycle of heme degradation.These findings suggest that in addition to proton delivery to the nascent FeIII-OO- intermediate duringcatalysis, 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 polypeptidevia small rearrangements in the network of hydrogen bonds and (ii) modulate the conformational freedomof the enzyme, thus allowing it to adapt to the demanding changes in axial coordination state and substratetransformations 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 R80Lmutant is likely related to its significantly lower efficiency to hydroxylate heme in the presence of H2O2,relative to the wild type enzyme.