Klein M L, Fulco A J
Department of Biological Chemistry, UCLA School of Medicine.
Biochim Biophys Acta. 1994 Nov 11;1201(2):245-50. doi: 10.1016/0304-4165(94)90047-7.
Cytochrome P-450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase that resembles the Class II P-450 systems of the eukaryotic endoplasmic reticulum. Its single polypeptide chain contains both a P-450 heme domain and an NADPH:P-450 reductase domain, each of which bears significant structural and functional homology with its microsomal counterparts. We report here that cytochrome c, which can accept NADPH-derived electrons from the reductase domain of P-450-BM-3, did not inhibit myristate hydroxylation catalyzed by P-450BM-3 or by two reductase domain mutant enzymes (W574Y, W574F) which have diminished hydroxylase activity relative to wild-type enzyme but retain cytochrome c reductase activity levels comparable to wild-type enzyme. Because reduced cytochrome c generated independently of the reductase domain of P-450BM-3 did not support myristate hydroxylation, it seems likely that cytochrome c binds to a site on the reductase domain which does not overlap the site of the heme domain interaction. We also found that myristate did not inhibit P-450BM-3-mediated cytochrome c reduction. Since neither substrate inhibited the conversion of the other, we conclude that the rate-limiting steps for both myristate hydroxylation and cytochrome c reduction by P-450BM-3 do not involve electron transfer through the reductase domain.
来自巨大芽孢杆菌的细胞色素P-450BM-3是一种可溶性、催化自足的脂肪酸单加氧酶，类似于真核内质网的II类P-450系统。其单条多肽链包含一个P-450血红素结构域和一个NADPH：P-450还原酶结构域，每个结构域与其微粒体对应物在结构和功能上都具有显著的同源性。我们在此报告，细胞色素c可从P-450-BM-3的还原酶结构域接受NADPH衍生的电子，但它并不抑制P-450BM-3或两种还原酶结构域突变酶（W574Y、W574F）催化的肉豆蔻酸羟基化反应。相对于野生型酶，这两种突变酶的羟化酶活性降低，但保留了与野生型酶相当的细胞色素c还原酶活性水平。由于独立于P-450BM-3还原酶结构域产生的还原型细胞色素c不支持肉豆蔻酸羟基化反应，细胞色素c似乎很可能结合到还原酶结构域上一个与血红素结构域相互作用位点不重叠的位点。我们还发现肉豆蔻酸并不抑制P-450BM-3介导的细胞色素c还原。由于两种底物都不抑制另一种底物的转化，我们得出结论，P-450BM-3催化肉豆蔻酸羟基化和细胞色素c还原的限速步骤不涉及通过还原酶结构域的电子转移。
