Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 2009 Dec 29;48(51):12113-24. doi: 10.1021/bi901187u.
The R481 residue of cytochrome bo(3) ubiquinol oxidase from E. coli is highly conserved in the heme-copper oxidase superfamily. It has been postulated to serve as part of a proton loading site that regulates proton translocation across the protein matrix of the enzyme. Along these lines, proton pumping efficiency has been demonstrated to be abolished in many R481 mutants. However, R481Q in bo(3) from E. coli has been shown to be fully functional, implying that the positive charge of the arginine is not required for proton translocation [ Puustinen , A. and Wikstrom , M. ( 1999 ) Proc. Natl. Acad. Sci. U.S.A. 96 , 35 - 37 ]. In an effort to delineate the structural role of R481 in the bo(3) oxidase, we used resonance Raman spectroscopy to compare the nonfunctional R481L mutant and the functional R481Q mutant, to the wild type protein. Resonance Raman data of the oxidized and reduced forms of the R481L mutant indicate that the mutation introduces changes to the heme o(3) coordination state, reflecting a change in position and/or coordination of the Cu(B) located on the distal side of heme o(3), although it is approximately 10 A away from R481. In the reduced-CO adduct of R481L, the frequencies of the Fe-CO and C-O stretching modes indicate that, unlike the wild type protein, the Cu(B) is no longer close to the heme-bound CO. In contrast, resonance Raman data obtained from the various oxidation and ligation states of the R481Q mutant are similar to those of the wild type protein, except that the mutation causes an enhancement of the relative intensity of the beta conformer of the CO-adduct, indicating a shift in the equilibrium between the alpha and beta conformers. The current findings, together with crystallographic structural data of heme-copper oxidases, indicate that R481 plays a keystone role in stabilizing the functional structure of the Cu(B) site through a hydrogen bonding network involving ordered water molecules. The implications of these data on the proton translocation mechanism are considered.
大肠杆菌细胞色素 bo(3)泛醌氧化还原酶的 R481 残基在血红素铜氧化酶超家族中高度保守。据推测，它充当质子加载位点的一部分，调节酶蛋白基质中的质子转运。沿着这些思路，已经证明许多 R481 突变体的质子泵效率被废除。然而，已经表明大肠杆菌 bo(3)中的 R481Q 是完全功能的，这意味着精氨酸的正电荷对于质子转运不是必需的[Puustinen，A.和 Wikstrom，M.（1999）Proc. Natl. Acad. Sci. U.S.A. 96，35-37]。为了描绘 R481 在 bo(3)氧化酶中的结构作用，我们使用共振拉曼光谱法比较了非功能 R481L 突变体和功能 R481Q 突变体与野生型蛋白。R481L 突变体氧化和还原形式的共振拉曼数据表明，突变导致血红素 o(3)配位状态发生变化，反映出位于血红素 o(3)远端的 Cu(B)的位置和/或配位发生变化，尽管它距离 R481 约 10 Å。在 R481L 的还原-CO 加合物中，Fe-CO 和 C-O 伸缩模式的频率表明，与野生型蛋白不同，Cu(B)不再靠近血红素结合的 CO。相比之下，从 R481Q 突变体的各种氧化和配位状态获得的共振拉曼数据与野生型蛋白相似，除了突变导致 CO 加合物的β构象的相对强度增强，表明α和β构象之间的平衡发生了转移。当前的发现，以及血红素铜氧化酶的晶体结构数据，表明 R481 通过涉及有序水分子的氢键网络在稳定 Cu(B)位点的功能结构方面发挥关键作用。考虑了这些数据对质子转运机制的影响。
