Sequential NMR assignment of the ferri-cytochrome c3 from Desulfovibrio vulgaris Hildenborough.

Abstract

Cytochromes c3 are low redox potential cytochromes involved in anaerobic metabolism. These periplasmic proteins contain four bi-histidinyl coordinated hemes. Multiheme cytochromes have been found in sulfate reducing bacteria of the genus Desulfovibrio. Preliminary analysis of Desulfovibrio vulgaris Hildenborough genome pointed out the existence of several putative genes encoding tetraheme cytochromes (http://www.tigr.org). Analysis of their genomic context showed that some of them are isolated (i.e., the gene encoding the well known soluble cytochrome c3 (Mr 13,000)) while others are part of multienzymatic complexes (i.e., the tetraheme cytochrome subunit in formate dehydrogenase (Sebban et al., 1995)). The large diversity of the tetraheme cytochromes in this organism must be correlated with the great specificity of these molecules for their oxidoreduction partners. We have recently reported a new approach to study electron transfer complexes combining NMR spectroscopy and theoretical calculations. 1H-15N HSQC are performed on an 15N-labelled redox partner, and we use the chemical shift variations induced upon complex formation to map the interacting site and to filter the ab initio models obtained by Bigger (Morelli et al., 2000). 1H-15N HSQC assignment is thus the first step of the functional study of cytochromes c3. We have initiated our studies with the soluble cytochrome c3 (Mr 13,000). The gene of this protein was cloned and sequenced (Voordouw et al., 1985) and the structure of this tetraheme cytochrome was solved by x-ray (Matias et al., 1993).