Madeo Jennifer, Gunner M R
Physics Department J-419 City College of New York 138th Street and Convent Avenue, New York, New York 10031, USA.
Biochemistry. 2005 Aug 23;44(33):10994-1004. doi: 10.1021/bi050544j.
Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentration and solvent viscosity dependence of the association rate indicate a second-order rate-determining step. The association rate constants (k(on)) range from 10(5) to 10(7) M(-)(1) s(-)(1). Association and dissociation rate constants were determined at pH values above the hydroxyl pK(a) for five hydroxyl naphthoquinones. These negatively charged compounds are competitive inhibitors for the Q(A) site. While the neutral quinones reach equilibrium in milliseconds, anionic hydroxyl quinones with similar K(d) values take minutes to bind or dissociate. These slow rates are independent of ionic strength, solvent viscosity, and quinone concentration, indicating a first-order rate-limiting step. The anionic semiquinone, formed by forward electron transfer at the Q(A) site, also dissociates slowly. It is not possible to measure the association rate of the unstable semiquinone. However, as the protein creates kinetic barriers for binding and releasing anionic hydroxyl quinones without greatly increasing the affinity relative to neutral quinones, it is suggested that the Q(A) site may do the same for anionic semiquinone. Thus, the slow semiquinone dissociation may not indicate significant thermodynamic stabilization of the reduced species in the Q(A) site.
细菌反应中心（RCs）催化一系列电子转移反应，将中性醌还原为结合态的阴离子半醌。测量并比较了13种无尾中性和阴离子型苯醌及萘醌与Q(A)位点的解离常数和缔合速率。这些醌的K(d)值范围为0.08至90微摩尔。对于包括杜醌（DQ）和2,3 - 二甲氧基 - 5 - 甲基 - 1,4 - 苯醌（UQ(0)）在内的8种中性醌，缔合速率对醌浓度和溶剂粘度的依赖性表明存在二级速率决定步骤。缔合速率常数（k(on)）范围为10(5)至10(7) M(-)(1) s(-)(1)。在高于5种羟基萘醌羟基pK(a)的pH值下测定了缔合和解离速率常数。这些带负电荷的化合物是Q(A)位点的竞争性抑制剂。虽然中性醌在几毫秒内达到平衡，但具有相似K(d)值的阴离子羟基醌结合或解离需要几分钟。这些缓慢的速率与离子强度、溶剂粘度和醌浓度无关，表明存在一级速率限制步骤。通过在Q(A)位点进行正向电子转移形成的阴离子半醌解离也很缓慢。无法测量不稳定半醌的缔合速率。然而，由于蛋白质在不相对于中性醌大幅增加亲和力的情况下为结合和释放阴离子羟基醌创造了动力学障碍，因此有人提出Q(A)位点对阴离子半醌可能也会如此。因此，缓慢的半醌解离可能并不表明Q(A)位点中还原态物种具有显著的热力学稳定性。
