Aagaard A, Gilderson G, Gomes C M, Teixeira M, Brzezinski P
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Biochemistry. 1999 Aug 3;38(31):10032-41. doi: 10.1021/bi990473m.
We have investigated the kinetic and thermodynamic properties of carbon monoxide binding to the fully reduced quinol oxidase (cytochrome aa(3)) from the hyperthermophilic archaeon Acidianus ambivalens. After flash photolysis of CO from heme a(3), the complex recombines with an apparent rate constant of approximately 3 s(-1), which is much slower than with the bovine cytochrome c oxidase (approximately 80 s(-1)). Investigation of the CO-recombination rate as a function of the CO concentration shows that the rate saturates at high CO concentrations, which indicates that CO must bind transiently to Cu(B) before binding to heme a(3). With the A. ambivalens enzyme the rate reached 50% of its maximum level (which reflects the dissociation constant of the Cu(B)(CO) complex) at approximately 13 microM CO, which is a concentration approximately 10(3) times smaller than for the bovine enzyme (approximately 11 mM). After CO dissociation we observed a rapid absorbance relaxation with a rate constant of approximately 1.4 x 10(4) s(-1), tentatively ascribed to a heme-pocket relaxation associated with release of CO after transient binding to Cu(B). The equilibrium constant for CO transfer from Cu(B) to heme a(3) was approximately 10(4) times smaller for the A. ambivalens than for the bovine enzyme. The approximately 10(3) times smaller Cu(B)(CO) dissociation constant, in combination with the approximately 10(4) times smaller equilibrium constant for the internal CO transfer, results in an apparent dissociation constant of the heme a(3)(CO) complex which is "only" about 10 times larger for the A. ambivalens ( approximately 4 x 10(-3) mM) than for the bovine (0.3 x 10(-3) mM) enzyme. In summary, the results show that while the basic mechanism of CO binding to the binuclear center is similar in the A. ambivalens and bovine (and R. sphaeroides) enzymes, the heme-pocket dynamics of the two enzymes are dramatically different, which is discussed in terms of the different structural details of the A. ambivalens quinol oxidase and adaptation to different living conditions.
我们研究了一氧化碳与嗜热古菌嗜酸两面菌（Acidianus ambivalens）完全还原的喹啉氧化酶（细胞色素aa(3)）结合的动力学和热力学性质。从血红素a(3)上进行一氧化碳的闪光光解后，该复合物以约3 s⁻¹的表观速率常数重新结合，这比牛细胞色素c氧化酶（约80 s⁻¹）慢得多。对一氧化碳再结合速率作为一氧化碳浓度的函数进行研究表明，在高一氧化碳浓度下速率达到饱和，这表明一氧化碳在与血红素a(3)结合之前必须先与Cu(B)短暂结合。对于嗜酸两面菌的酶，在约13 μM一氧化碳时速率达到其最大水平的50%（这反映了Cu(B)(CO)复合物的解离常数），该浓度比牛酶（约11 mM）小约10³倍。一氧化碳解离后，我们观察到快速的吸光度弛豫，速率常数约为1.4×10⁴ s⁻¹，初步归因于与一氧化碳在短暂结合到Cu(B)后释放相关的血红素口袋弛豫。对于嗜酸两面菌，一氧化碳从Cu(B)转移到血红素a(3)的平衡常数比牛酶小约10⁴倍。约小10³倍的Cu(B)(CO)解离常数，与约小10⁴倍的内部一氧化碳转移平衡常数相结合，导致血红素a(3)(CO)复合物的表观解离常数对于嗜酸两面菌（约4×10⁻³ mM）“仅”比牛酶（0.3×10⁻³ mM）大约10倍。总之，结果表明，虽然一氧化碳与双核中心结合的基本机制在嗜酸两面菌和牛（以及球形红杆菌（R. sphaeroides））的酶中相似，但这两种酶的血红素口袋动力学却有显著差异，这将根据嗜酸两面菌喹啉氧化酶的不同结构细节以及对不同生活条件的适应性进行讨论。
