Purification and properties of a novel cytochrome: flavocytochrome c from Shewanella putrefaciens

Abstract

The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.

Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c required for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1

“…Specific fumarate reductase activities were 20-to 30-fold higher in the soluble fraction than in the other fractions (Table 3), with Ͼ98% of the total fumarate reductase activity in the soluble fraction. This is consistent with previous findings (35,38) and the periplasmic location of this enzyme (31,35,48). Analysis for membrane buoyant density and SDS-PAGE patterns (Fig.…”

“…Consistent with this, a typical bacterial secretory signal sequence (61) or signal peptidase cleavage site (60) could not be identified in CymA. However, since there is only one reported example of a secretory signal sequence for a periplasmic protein in S. putrefaciens (31,48), further studies will be necessary to definitively address this issue with CymA.…”

“…MR-1 contains numerous respiratory electron transport components, including three quinones (ubiquinone, menaquinone, and methylmenaquinone) (38), and several c-and b-type cytochromes localized in various subcellular regions (36). Previous reports have demonstrated key differences other than its ability to utilize insoluble substrates (e.g., Mn and Fe oxides) as electron acceptors between the electron transport chains of MR-1 and those of other bacteria: (i) MR-1 localizes a majority of its membrane-bound cytochromes to its OM when grown under anaerobic conditions (36), (ii) the fumarate reductase of MR-1 is a periplasmic enzyme (31,35,48) in contrast to the membrane-bound forms in other bacteria, and (iii) in direct contrast to E. coli, menaquinone is required for nitrate reduction in MR-1 but is not required for TMAO reduction (38).…”

Extracellular respiration of dimethyl sulfoxide by Shewanella oneidensis strain MR-1

“…This resistance was exhibited both in whole cells and in cell lysates. As a control, fumarate reductase activity [a periplasmic enzyme in S. oneidensis (29)] was monitored with and without protease with both whole cells and lysates ( Table 1). As expected, 40% of the fumarate reductase activity was eliminated in cell lysates after 1 h, yet DMSO reductase activity was unchanged (Table 1), even after 5 h of protease treatment (data not shown).…”

Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens

“…In all of the above-cited studies, cytochrome content and type were judged mainly from spectral data, using the intensity of the Soret band after treatment with dithionite, or by heme staining polyacrylamide gels with tetramethylbenzidine (18). In recent and more definitive studies, a large tetraheme flavocytochrome c fumarate reductase was purified from S. putrefaciens (NCIMB400), and the gene sequence was determined (6,13). This enzyme is a 63.8-kDa soluble protein, and unlike the usual membrane-bound fumarate reductase, it contains heme c instead of iron-sulfur centers, and the heme midpoint redox potentials of Ϫ220 and Ϫ320 mV are unusually low.…”