Wang Jinling, Evans John P, Ogura Hiroshi, La Mar Gerd N, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, California 94143-2280, USA.
Biochemistry. 2006 Jan 10;45(1):61-73. doi: 10.1021/bi051645k.
Heme oxygenase regiospecifically oxidizes heme at the alpha-meso position to give biliverdin IXalpha, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry but partially shifts the oxidation to the beta/delta-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by approximately 90 degrees, causes a slight loss of regiospecificity but combined with the R183E and K18E mutations results primarily in beta/delta-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane.
血红素加氧酶在α-中位区域特异性地氧化血红素，生成胆绿素IXα、一氧化碳和铁。活性位点内的血红素取向被认为决定了氧化区域特异性，此处显示人源酶（hHO1）的血红素取向在很大程度上由血红素羧酸基团与精氨酸183和赖氨酸18之间的相互作用决定，而非酪氨酸134。单独突变精氨酸183或赖氨酸18不会显著改变依赖于NADPH-细胞色素P450还原酶的反应区域化学，但在抗坏血酸支持的反应中会使氧化部分转移至β/δ-中位位置。将谷氨酸29突变为赖氨酸，若血红素旋转约90度，该正电荷可与血红素羧基相互作用，这会导致区域特异性略有丧失，但与R183E和K18E突变结合后，在所有条件下主要导致血红素的β/δ-中位氧化。对血红素与三重K18E/E29K/R183E突变体结合的NMR分析证实了活性位点内血红素的旋转。动力学研究表明，精氨酸183的突变极大地损害了依赖于P450还原酶的反应速率，这与早期发现精氨酸183参与还原酶与hHO1的结合一致，但对抗坏血酸反应影响很小。破坏活性位点氢键网络的天冬氨酸140和酪氨酸58的突变会损害催化速率，但不影响氧化区域化学。结果表明，氧化区域化学在很大程度上由血红素丙酸基团与蛋白质的离子相互作用控制，区域特异性的改变涉及血红素围绕垂直于血红素平面的轴的旋转。
