Unbinding of oxidized cytochrome c from photosynthetic reaction center of Rhodobacter sphaeroides is the bottleneck of fast turnover
Unbinding of oxidized cytochrome c from photosynthetic reaction center of Rhodobacter sphaeroides is the bottleneck of fast turnover
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DOI：
10.1021/bi991563u
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发表时间：
1999-12-21
期刊：
影响因子：
2.9
通讯作者：
Maróti, P
中科院分区：
文献类型：
--
作者：
Gerencsér, L;Laczkó, G;Maróti, P
为了了解光合反应中心周转速率限制的细节，在各种细胞色素浓度、离子强度、粘度、温度、光强度和 pH 条件下，通过强连续照明检查了洗涤剂分散体中的球状红杆菌反应中心对马心细胞色素 c 的光氧化作用。观察到的细胞色素稳态周转率不受光强度限制。根据最近的发现 [Larson, J. W.、Wells, T. A. 和 Wraight, C. A. (1998) Biophys. J. 74 (2), A76]，在 0-40 mM NaCl 范围内，从 1000 到 2300 s(-1)，周转率随着体积离子强度的增加而增加，然后在细胞色素和泛醌过量以及光化学速率常数为 4500 s(-1) 的条件下，在高离子强度下周转率下降。此外，我们发现以下内容：（i）供体（细胞色素 c）和受体（泛醌）侧的贡献以及还原细胞色素 c 的结合和氧化细胞色素 c 的释放可以在观察到的动力学中分开。在中性和酸性 pH 值（当质子转移不受速率限制时）和低或中等离子强度下，反应中心的周转速率主要受到光氧化细胞色素 c 的低释放速率（产物抑制）的限制。然而，在高离子强度下，还原细胞色素c的结合率急剧下降，成为瓶颈。观察到的稳态周转率活化能反映了限制机制的变化：4 mM 离子强度时为 1.5 kcal/mol，100 mM 离子强度时为 5.7 kcal/mol。在周转率的粘度依赖性中也观察到类似的区别：在 40 和 100 mM 离子强度下，下降幅度较大 (eta(-1))，在低盐 (4 mM) 条件下下降幅度适中 (eta(-0.2))。 (ii)受体侧与过量泛醌-30或泛醌-50的醌交换率高于供体侧的细胞色素交换，并且不限制观察到的细胞色素周转率。 (iii) 多价阳离子不仅通过离子强度（筛选）发挥作用，还通过与表面电荷基团的直接相互作用（离子对产生）发挥作用。重金属离子 Cd2+ 与 RC 结合，表观解离常数为 14 μM。 (iv) 反应中心和细胞色素 c 之间的碰撞相互作用的双态模型以及计算速率常数时的简单静电考虑通常足以描述二聚体和细胞色素 c 的光氧化动力学。 (v) 细胞色素周转率的 pH 依赖性表明，高光条件下细胞色素的稳态周转率不是由反应中心的等电点 (pI = 6.1) 决定的，而是由对接位点附近的羧基残基决定的。
To understand the details of rate limitation of turnover of the photosynthetic reaction center, photooxidation of horse heart cytochrome c by reaction center from Rhodobacter spheroides in detergent dispersion has been examined by intense continuous illumination under a wide variety of conditions of cytochrome concentration, ionic strength, viscosity, temperature, light intensity, and pH. The observed steady-state turnover rate of the cytochrome was not Light intensity limited. In accordance with recent findings [Larson, J. W., Wells, T. A., and Wraight, C. A. (1998) Biophys. J. 74 (2), A76], the turnover rate increased with increasing bulk ionic strength in the range of 0-40 mM NaCl from 1000 up to 2300 s(-1) and then decreased at high ionic strength under conditions of excess cytochrome and ubiquinone and a photochemical rate constant of 4500 s(-1). Furthermore, we found the following: (i) The contribution of donor (cytochrome c) and acceptor (ubiquinone) sides as well as the binding of reduced and the release of oxidized cytochrome c could be separated in the observed kinetics. At neutral and acidic pH (when the proton transfer is not rate limiting) and at low or moderate ionic strength, the turnover rate of the reaction center was limited primarily by the low release rate of the photooxidized cytochrome c (product inhibition). At high ionic strength, however, the binding rate of the reduced cytochrome c decreased dramatically and became the bottleneck. The observed activation energy of the steady-state turnover rate reflected the changes in limiting mechanisms: 1.5 kcal/mol at 4 mM and 5.7 kcal/mol at 100 mM ionic strength. A similar distinction was observed in the viscosity dependence of the turnover rate: the decrease was steep (eta(-1)) at 40 and 100 mM ionic strengths and moderate (eta(-0.2)) under low-salt (4 mM) conditions. (ii) The rate of quinone exchange at the acceptor side with excess ubiquinone-30 or ubiquinone-50 was higher than the cytochrome exchange at the donor side and did not limit the observed rate of cytochrome turnover. (iii) Multivalent cations exerted effects not only through ionic strength (screening) but also by direct interaction with surface charge groups (ion-pair production). Heavy metal ion Cd2+ bound to the RC with apparent dissociation constant of 14 mu M. (iv) A two-stale model of collisional interaction between reaction center and cytochrome c together with simple electrostatic considerations in the calculation of rate constants was generally sufficient to describe the kinetics of photooxidation of dimer and cytochrome c. (v) The pH dependence of cytochrome turnover rate indicated that the steady-state turnover rate of the cytochrome under high light conditions was not determined by the isoelectric point of the reaction center (pI = 6.1) but by the carboxyl residues near the docking site.