Nagano Shingo, Shimada Hideo, Tarumi Akiko, Hishiki Takako, Kimata-Ariga Yoko, Egawa Tsuyoshi, Suematsu Makoto, Park Sam-Yong, Adachi Shin-ichi, Shiro Yoshitsugu, Ishimura Yuzuru
Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan.
Biochemistry. 2003 Dec 16;42(49):14507-14. doi: 10.1021/bi035410p.
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.
细胞色素P450cam的亚铁 - 一氧化碳结合形式（CO - P450cam）在1940和1932 cm⁻¹处有两个红外（IR）CO伸缩带。对于不含恶臭假单胞菌铁氧还蛋白（Pdx）的CO - P450cam，前一个带占主导（面积>95%），而在CO - P450cam与还原型Pdx的复合物中，后一个带占主导（面积>95%）。因此，Pdx与CO - P450cam的结合引起了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，将P450cam中的表面氨基酸残基Arg79、Arg109和Arg112替换为Lys、Gln和Met。对突变体的红外光谱和动力学分析表明，在结合Pdx后具有较大1932 cm⁻¹带面积的酶具有较大的催化活性。对R109K和R112K晶体结构的研究表明，Arg112的胍基与Pdx之间的相互作用对于构象变化很重要。这些突变并没有改变羟基化产物与消耗的氧气之间的偶联比。我们将这些发现解释为意味着P450cam与Pdx通过Arg112的相互作用增强了从近端配体（Cys357）到铁结合的O₂的O - O键的电子供体作用，并且可能促进了从还原型Pdx到氧合P450cam的电子转移，从而促进了O - O键的断裂。
