Chang S L, Wallar B J, Lipscomb J D, Mayo K H
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis 55455, USA.
Biochemistry. 2001 Aug 14;40(32):9539-51. doi: 10.1021/bi0103462.
Methane monooxygenase (MMO) is a non-heme-iron-containing enzyme which consists of 3 protein components: a hydroxylase (MMOH), an NAD(P)H-linked reductase (MMOR), and a 138-residue regulatory protein, component B (MMOB). Here, NMR spectroscopy has been used to derive interactions between MMOB and reduced and oxidized states of MMOH (245 kDa). Differential broadening of MMOB resonances in 1H-15N HSQC spectra acquired at different molar ratios of MMOH indicates interaction of both proteins, with MMOB binding more tightly to oxidized MMOH as observed previously. The most broadened backbone NH resonances suggest which residues in MMOB are part of the MMOH-binding interface, particularly when those residues are spatially close or clustered in the structure of MMOB. Although a number of different residues in MMOB appear to be involved in interacting with oxidized- and reduced-MMOH, some are identical. The two most common segments, proximal in the structure of MMOB, are beta-strand 1 with turn 1 (residues 36-46) and alpha-helix 3 going into loop 2 (residues 101-112). In addition, the N-terminus of MMOB is observed to be involved in binding to MMOH in either redox state. This is most strongly evidenced by use of a synthetic N-terminal peptide from MMOB (residues 1-29) in differential broadening 1H TOCSY studies with MMOH. Binding specificity is demonstrated by displacement of the peptide from MMOH by parent MMOB, indicating that the peptide binds in or near the normal site of N-terminal binding. The N-terminus is also observed to be functionally important. Steady-state kinetic studies show that neither a delta2-29 MMOB deletion mutant (which in fact does bind to MMOH), the N-terminal peptide, nor a combination of the two elicit the effector functions of MMOB. Furthermore, transient kinetic studies indicate that none of the intermediates of the MMOH catalytic cycle are observed if either the delta2-29 MMOB mutant or the N-terminal peptide is used in place of MMOB, suggesting that deletion of the N-terminus prevents reaction of reduced MMOH with O2 that initiates catalysis.
甲烷单加氧酶（MMO）是一种不含血红素铁的酶，由3种蛋白质成分组成：一种羟化酶（MMOH）、一种与NAD（P）H相连的还原酶（MMOR），以及一种由138个残基组成的调节蛋白B组分（MMOB）。在此，核磁共振光谱已被用于推导MMOB与MMOH（245 kDa）的还原态和氧化态之间的相互作用。在不同MMOH摩尔比下获得的1H-15N HSQC光谱中，MMOB共振峰的差异展宽表明两种蛋白质之间存在相互作用，正如之前所观察到的，MMOB与氧化态的MMOH结合更紧密。展宽最明显的主链NH共振峰表明MMOB中的哪些残基是MMOH结合界面的一部分，特别是当这些残基在MMOB结构中空间上接近或聚集时。虽然MMOB中许多不同的残基似乎都参与了与氧化态和还原态MMOH的相互作用，但有些是相同的。在MMOB结构中靠近的两个最常见片段是带有转角1的β链1（残基36 - 46）和进入环2的α螺旋3（残基101 - 112）。此外，观察到MMOB的N末端在两种氧化还原状态下均参与与MMOH的结合。在与MMOH进行的差异展宽1H TOCSY研究中使用来自MMOB的合成N末端肽（残基1 - 29），最有力地证明了这一点。母体MMOB能将该肽从MMOH上置换下来，证明了结合特异性，表明该肽在N末端结合的正常位点或其附近结合。还观察到N末端在功能上很重要。稳态动力学研究表明，δ2 - 29 MMOB缺失突变体（实际上它确实能与MMOH结合）、N末端肽，或两者的组合都不会引发MMOB的效应功能。此外，瞬态动力学研究表明，如果使用δ2 - 29 MMOB突变体或N末端肽代替MMOB，则观察不到MMOH催化循环的任何中间体，这表明N末端的缺失会阻止还原态的MMOH与启动催化作用的O2发生反应。
