Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam
Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam
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DOI：
10.1021/bi035410p
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发表时间：
2003-12-16
期刊：
影响因子：
2.9
通讯作者：
Ishimura, Y
中科院分区：
文献类型：
--
作者：
Nagano, S;Shimada, H;Ishimura, Y
细胞色素 P450cam (CO-P450cam) 的亚铁-一氧化碳结合形式在 1940 和 1932 cm(-1) 处有两个红外 (IR) CO 伸缩带。对于不含恶臭氧还蛋白 (Pdx) 的 CO-P450cam，前一条带占主导地位（面积 >95%），而在 Pdx 减少的 CO-P450cam 复合物中，后一条带占主导地位（面积>95%）。因此，Pdx 与 CO-P450cam 的结合引起血红素活性位点的构象变化。为了研究构象变化所涉及的机制，P450cam 中的表面氨基酸残基 Arg79、Arg109 和 Arg112 被替换为 Lys、Gln 和 Met。对突变体的红外光谱和动力学分析表明，与Pdx结合时具有较大1932 cm(-1)带面积的酶具有较大的催化活性。对 R109K 和 R112K 晶体结构的检查表明，Arg112 的胍基和 Pdx 之间的相互作用对于构象变化很重要。突变没有改变羟基化产物和消耗的氧气之间的耦合比。我们将这些发现解释为意味着 P450cam 通过 Arg112 与 Pdx 的相互作用增强了从近端配体 (Cys357) 到铁结合 O-2 的 O-O 键的电子供给，并且可能促进电子从还原的 Pdx 转移到 oxyP450cam，从而促进 O-O 键分裂。
Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O-2 and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.