Takayama Shin-Ichi J, Loutet Slade A, Mauk A Grant, Murphy Michael E P
†Department of Microbiology and Immunology, ‡Department of Biochemistry and Molecular Biology, and §UBC Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Biochemistry. 2015 Apr 28;54(16):2613-21. doi: 10.1021/acs.biochem.5b00239. Epub 2015 Apr 16.
The canonical heme oxygenases (HOs) catalyze heme oxidation via a heme-bound hydroperoxo intermediate that is stabilized by a water cluster at the active site of the enzyme. In contrast, the hydrophobic active site of IsdI, a heme-degrading enzyme from Staphylococcus aureus, lacks a water cluster and is expected to oxidize heme by an alternative mechanism. Reaction of the IsdI-heme complex with either H2O2 or m-chloroperoxybenzoic acid fails to produce a specific oxidized heme iron intermediate, suggesting that ferric-hydroperoxo or ferryl derivatives of IsdI are not involved in the catalytic mechanism of this enzyme. IsdI lacks a proton-donating group in the distal heme pocket, so the possible involvement of a ferric-peroxo intermediate has been evaluated. Density functional theory (DFT) calculations indicate that heme oxidation involving a ferric-peroxo intermediate is energetically accessible, whereas the energy barrier for a reaction involving a ferric-hydroperoxo intermediate is too great in the absence of a proton donor. We propose that IsdI catalyzes heme oxidation through nucleophilic attack by the heme-bound peroxo species. This proposal is consistent with our previous demonstration by nuclear magnetic resonance spectroscopy that heme ruffling increases the susceptibility of the meso-carbon of heme to nucleophilic attack.
典型的血红素加氧酶（HOs）通过与血红素结合的氢过氧中间体催化血红素氧化，该中间体由酶活性位点处的水簇稳定。相比之下，金黄色葡萄球菌的血红素降解酶IsdI的疏水活性位点缺乏水簇，预计会通过另一种机制氧化血红素。IsdI-血红素复合物与过氧化氢或间氯过氧苯甲酸反应均未能产生特定的氧化血红素铁中间体，这表明IsdI的铁-氢过氧或高铁酰基衍生物不参与该酶的催化机制。IsdI在血红素远端口袋中缺乏质子供体基团，因此对铁-过氧中间体的可能参与情况进行了评估。密度泛函理论（DFT）计算表明，涉及铁-过氧中间体的血红素氧化在能量上是可行的，而在没有质子供体的情况下，涉及铁-氢过氧中间体的反应的能量屏障过大。我们提出IsdI通过与血红素结合的过氧物种的亲核攻击催化血红素氧化。这一提议与我们之前通过核磁共振光谱证明的血红素褶皱增加血红素中碳对亲核攻击的敏感性一致。
