A Biochemical Deconstruction-Based Strategy to Assist the Characterization of Bacterial Electric Conductive Filaments

Silva, Marta A.; Fernandes, Ana P.; Turner, David L.; Salgueiro, Carlos A.

http://hdl.handle.net/10362/157224

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Resumo(s)

Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes.

Descrição

The NMR spectrometers are part of the National NMR Network (PT NMR) and are supported by FCTMCTES (ROTEIRO/0031/2013–PINFRA/22161/2016) co-funded by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC. Publisher Copyright: © 2023 by the authors.

Palavras-chave

electron transfer Geobacter multiheme cytochromes nanowires NMR Catalysis Molecular Biology Spectroscopy Computer Science Applications Physical and Theoretical Chemistry Organic Chemistry Inorganic Chemistry