Zhang Haoming, Gruenke Larry, Arscott Dave, Shen Anna, Kasper Charles, Harris Danni L, Glavanovich Michael, Johnson Richard, Waskell Lucy
University of Michigan and VA Medical Research Center, 2215 Fuller Road, Ann Arbor, Michigan 48105, USA.
Biochemistry. 2003 Oct 14;42(40):11594-603. doi: 10.1021/bi034968u.
The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH(2), while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s(-1) at 15 degrees C. Surprisingly, oxyferrous cyt P450 turns over more slowly with a rate constant of 0.09 s(-1), which is the rate of catalysis under steady-state conditions at 15 degrees C (k(cat) = 0.08 s(-1)). In contrast, the rate constant for electron transfer from ferrous cyt b(5) to oxyferrous cyt P450 is 10 s(-1) with oxyferrous cyt P450 and cyt b(5) simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b(5) and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b(5) and that the rate-limiting step in catalysis follows introduction of the second electron.
使用5-脱氮黄素腺嘌呤二核苷酸（5-deazaFAD）T491V细胞色素P450还原酶使得直接测量电子传递至微粒体氧合亚铁细胞色素（cyt）P450 2B4的速率成为可能。在这种还原酶中，黄素单核苷酸（FMN）部分可被还原为氢醌（FMNH₂），而5-脱氮黄素腺嘌呤二核苷酸部分仍保持氧化状态[Zhang, H., 等人（2003年）《生物化学》42, 6804 - 6813]。在底物存在的情况下，通过将亚铁细胞色素P450 - 2电子还原的5-脱氮黄素腺嘌呤二核苷酸T491V还原酶复合物与氧气快速混合，测定了从5-脱氮黄素腺嘌呤二核苷酸细胞色素P450还原酶到氧合亚铁细胞色素P450的电子传递速率。只能提供单个电子的5-脱氮黄素腺嘌呤二核苷酸T491V还原酶将氧合亚铁细胞色素P450还原，并氧化为空气稳定的半醌，在15℃下的速率常数分别为8.4和0.37 s⁻¹。令人惊讶的是，氧合亚铁细胞色素P450的周转更慢，速率常数为0.09 s⁻¹，这是15℃稳态条件下的催化速率（kcat = 0.08 s⁻¹）。相比之下，从亚铁细胞色素b5到氧合亚铁细胞色素P450的电子传递速率常数为10 s⁻¹，同时氧合亚铁细胞色素P450和细胞色素b5会发生光谱变化。液相色谱 - 串联质谱（LC-MS/MS）的定量分析表明，产物去甲苄非他明的形成与细胞色素b5的偶联效率为52%，与5-脱氮黄素腺嘌呤二核苷酸T491V还原酶的偶联效率为32%。总体而言，这些结果表明，在催化过程中，在细胞色素P450还原酶存在下会形成相对稳定的细胞色素P450还原型氧合亚铁中间体，但在细胞色素b5存在下则不会，并且催化过程中的限速步骤发生在第二个电子引入之后。
