Dantas Joana M, Kokhan Oleksandr, Pokkuluri P Raj, Salgueiro Carlos A
UCIBIO-Requimte, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal.
Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA 22807, USA.
Biochim Biophys Acta. 2015 Oct;1847(10):1129-38. doi: 10.1016/j.bbabio.2015.06.004. Epub 2015 Jun 9.
Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the HS analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for HS respiration in G. sulfurreducens, but the process is far from fully understood. In this work, NMR chemical shift perturbation measurements were used to map the interaction region between PpcA and AH2QDS, and to measure their binding affinity. The results showed that the AH2QDS binds reversibly to the more solvent exposed edge of PpcA heme IV. The NMR and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme IV (-127mV) compared to that of AH2QDS (-184mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between AH2QDS and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. Such behavior might confer selective advantage to G. sulfurreducens, which can utilize the HS in any redox state available in the environment for its metabolic needs.
腐殖质（HS）是陆地和水生环境中天然有机物质的重要组成部分，可作为厌氧微生物呼吸中的终端电子受体。硫还原地杆菌具有显著的呼吸多样性，能够利用HS类似物蒽醌 - 2,6 - 二磺酸盐（AQDS）作为终端电子受体，或其还原形式（AH2QDS）作为电子供体。先前的研究将三血红素细胞色素PpcA设定为硫还原地杆菌中HS呼吸的关键组分，但该过程远未被完全理解。在这项工作中，利用核磁共振化学位移扰动测量来绘制PpcA与AH2QDS之间的相互作用区域，并测量它们的结合亲和力。结果表明，AH2QDS可逆地结合到PpcA血红素IV更暴露于溶剂的边缘。结合氧化还原测量的核磁共振和可见光谱被用于确定PpcA：醌醇复合物的热力学参数。与AH2QDS（-184mV）相比，血红素IV（-127mV）具有更高的还原电位，这解释了为什么在醌醇还原细胞色素的情况下电子转移更有利。获得的关于AH2QDS与PpcA之间形成电子转移复合物的明确证据，与该蛋白质也与AQDS形成氧化还原复合物这一事实相结合，首次揭示了PpcA对HS类似物的双功能行为。这种行为可能赋予硫还原地杆菌选择性优势，使其能够利用环境中任何可用氧化还原状态的HS来满足其代谢需求。
