Paulsen K E, Liu Y, Fox B G, Lipscomb J D, Münck E, Stankovich M T
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Biochemistry. 1994 Jan 25;33(3):713-22. doi: 10.1021/bi00169a013.
Methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB) protein components. MMOH contains two oxygen-bridged dinuclear iron clusters that are the sites of O2 activation and hydrocarbon oxidation. Each cluster can be stabilized in diferric [Fe(III).Fc(III)], mixed-valence [Fe(II).Fe(III)], and diferrous [Fe(II).Fe(II)] redox states. We have correlated the EPR spin quantitation of the S = 1/2 mixed-valence state with the system electrode potential to determine both formal redox potential values for MMOH at 4 degrees C: E1 degrees' = +76 +/- 15 mV and E2 degrees' = +21 +/- 15 mV (Em = +48 mV, 61% maximum mixed-valence state). Complementary Mössbauer studies of 57Fe-enriched MMOH allowed all three redox states to be quantitated simultaneously in individual samples and revealed that the distribution of redox states was in accord with the measured potential values. EPR spectra of partially reduced MMOH showed that the apparent midpoint potential values of MMOH-MMOR, MMOH-MMOR-MMOB, and MMOH-MMOR-MMOB-substrate complexes were slightly more positive than that of MMOH alone. In contrast, the MMOH-MMOB complex appeared to have a substantially more negative redox potential. The formal redox potential values of the latter complex were determined to be E1 degrees' = -52 +/- 15 mV and E2 degrees' = -115 +/- 15 mV, respectively, at 4 degrees C (Em = -84 mV, 65% maximum mixed-valence state). This negative 132-mV shift in the midpoint potential of MMOH coupled to MMOB binding suggests that MMOB binds approximately 10(4) more strongly to the diferric state of MMOH than to the diferrous state. Since the potential shift is strongly negative, and since a nearly constant separation between the two formal potential values of MMOH is maintained when MMOB binds, the role of the MMOB-MMOH complex must not be to thermodynamically stabilize the formation of the diferrous cluster which is the form that reacts with O2 during catalysis. However, MMOB binding may provide kinetic stabilization of the diferrous state and/or modulation of the interaction of MMOH with O2 and hydrocarbon substrates. Such roles may be effected through cyclic association and dissociation of the MMOB-MMOH complex as MMOH oscillates between redox states during catalysis, thereby dynamically altering the affinity of this complex.
从甲基弯曲菌OB3b中分离出的甲烷单加氧酶（MMO）由羟化酶（MMOH）、还原酶（MMOR）和“B”蛋白组分（MMOB）组成。MMOH含有两个氧桥联双核铁簇，它们是O₂活化和烃氧化的位点。每个簇可以稳定在高铁[Fe(III).Fe(III)]、混合价态[Fe(II).Fe(III)]和亚铁[Fe(II).Fe(II)]氧化还原状态。我们已将S = 1/2混合价态的EPR自旋定量与系统电极电位相关联，以确定4℃下MMOH的两个形式氧化还原电位值：E1°' = +76 ± 15 mV和E2°' = +21 ± 15 mV（Em = +48 mV，最大混合价态为61%）。对富含⁵⁷Fe的MMOH进行的补充穆斯堡尔研究允许在单个样品中同时对所有三种氧化还原状态进行定量，并表明氧化还原状态的分布与测量的电位值一致。部分还原的MMOH的EPR光谱表明，MMOH-MMOR、MMOH-MMOR-MMOB和MMOH-MMOR-MMOB-底物复合物的表观中点电位值比单独的MMOH略正。相比之下，MMOH-MMOB复合物似乎具有实质上更负的氧化还原电位。在4℃下，后一种复合物的形式氧化还原电位值分别确定为E1°' = -52 ± 15 mV和E2°' = -115 ± 15 mV（Em = -84 mV，最大混合价态为65%）。与MMOB结合时MMOH中点电位的这种负132 mV偏移表明，MMOB与MMOH的高铁状态的结合比与亚铁状态的结合强约10⁴倍。由于电位偏移是强烈负的，并且由于当MMOB结合时MMOH的两个形式电位值之间保持几乎恒定间隔，因此MMOB-MMOH复合物的作用一定不是在热力学上稳定亚铁簇的形成，而亚铁簇是催化过程中与O₂反应的形式。然而，MMOB的结合可能提供亚铁状态的动力学稳定性和/或调节MMOH与O₂和烃底物的相互作用。当MMOH在催化过程中在氧化还原状态之间振荡时，这种作用可能通过MMOB-MMOH复合物的循环缔合和解离来实现，从而动态改变该复合物的亲和力。
