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Bioinorganic Chemistry and Applications
Volume 3 (2005), Issue 1-2, Pages 81-91
http://dx.doi.org/10.1155/BCA.2005.81

Purification and Preliminary Characterization of Tetraheme Cytochrome c3 and Adenylylsulfate Reductase from the Peptidolytic Sulfate-Reducing Bacterium Desulfovibrio aminophilus DSM 12254

1Centro de Investigaciones Biológicas del Noroeste, (CIBNOR). Mar Bermejo 195, Playa Palo Santa Rita, Baja California Sur, La Paz 23090, Mexico
2REQUIMTE/CQFB, Departamento de Quimica, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Monte de Caparica 2829-516, Portugal
3Laboratoire de Microbiologie IRD, UR 101, IFR-BAIM, ESIL Universités de Provence et de la Méditerranée, Campus de Luminy, Case 925, Marseille Cedex 09 13288, France

Copyright © 2005 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A278nm/A388nm) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c3 (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A553-A570]red/A280nm) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c3 from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence).