Pieulle Laetitia, Morelli Xavier, Gallice Philippe, Lojou Elisabeth, Barbier Pascale, Czjzek Mirjam, Bianco Pierre, Guerlesquin Françoise, Hatchikian E Claude
Unité de Bioénergétique et Ingéniérie des Protéines, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France.
J Mol Biol. 2005 Nov 18;354(1):73-90. doi: 10.1016/j.jmb.2005.09.036. Epub 2005 Sep 29.
In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.
在脱硫弧菌的代谢过程中，周质中的氢氧化作用通过跨膜电子传递复合物与细胞质中的硫酸盐还原作用相偶联。II型四血红素细胞色素c3（TpII-c3）、九血红素细胞色素c（9HcA）和十六血红素细胞色素c（HmcA）是与这些膜结合氧化还原复合物相关的周质蛋白，并表现出类似的生理功能。I型四血红素细胞色素c3（TpI-c3）被认为是从氢化酶向这些多血红素细胞色素进行电子传递的介质。在本研究中，我们研究了非洲脱硫弧菌（Da）和希登伯勒脱硫弧菌（DvH）的TpI-c3/TpII-c3复合物。利用电化学对Da TpI-c3和TpII-c3进行的比较动力学实验证实，作为氢化酶的电子受体，TpI-c3比TpII-c3效率高得多（二级速率常数k = 9×10⁸ M⁻¹ s⁻¹，Kₘ = 0.5 μM，而TpI-c3和TpII-c3的k分别为1.7×10⁷ M⁻¹ s⁻¹和Kₘ = 40 μM）。通过凝胶过滤、分析超速离心和交联实验，在低离子强度下对Da TpI-c3/TpII-c3复合物进行了表征。通过等温滴定量热法测定了热力学参数。复合物的形成主要由正的熵变驱动（ΔS = 137（±7）J mol⁻¹ K⁻¹，ΔH = 5.1（±1.3）kJ mol⁻¹），在pH 5.5时，缔合常数的值为（2.2（±0.5））×10⁶ M⁻¹。我们的热力学结果表明，焓和熵的净增加主要是由质子释放并伴随着水分子排除产生的。静电力在稳定两种蛋白质之间的复合物中起重要作用，因为在高离子强度下未检测到复合物形成。已以1.5埃的分辨率解析了Da TpI-c3的晶体结构，并通过核磁共振和对接实验获得了复合物的结构模型。对DvH TpI-c3/TpII-c3复合物进行了类似的实验。在这两种复合物中，TpI-c3的血红素IV面向TpII-c3的血红素I，涉及TpI-c3的碱性残基和TpII-c3的酸性残基。在这两种复合物中都观察到了一个二级相互作用位点，涉及Da TpII-c3的血红素II和DvH TpI-c3的血红素III，Da和DvH复合物的TpI-c3/TpII-c3摩尔比分别为2:1和1:2。讨论了多血红素细胞色素c中这些替代位点的生理意义。
