Overproduction of prismane protein in Desulfovibrio vulgaris (Hildenborough): evidence for a second S = 1/2-spin system in the one-electron reduced state.
The gene encoding the prismane protein from Desulfovibrio vulgaris (Hildenborough) was inserted into broad-host-range vector pSUP104. The recombinant plasmid, pJSP104, was transferred to D. vulgaris by conjugal plasmid transfer. In the transconjugant D. vulgaris cells the prismane protein was 25-fold overproduced. The overproduced prismane protein was characterized by molecular mass, isoelectric point, iron content and spectroscopical properties. Both the iron content and the ultraviolet/visible spectrum are identical to the wild-type protein indicating that iron incorporation in the overproduced protein is complete. EPR spectra of the dithionite-reduced form of the overproduced protein indicated that the Fe-S cluster might occur in a similar structure as found in inorganic model compounds containing a [6Fe-6S] prismane core. The as-isolated overproduced protein showed the presence of a second S = 1/2 spin system that was also detected in the corresponding prismane protein from D. desulfuricans (ATCC 27774), but not in the protein from wild-type D. vulgaris. This additional signal was irreversibly transformed to the 'wild-type' high-spin and low-spin systems upon two reduction/re-oxidation cycles. It is shown that the EPR spectroscopy of the overproduced prismane protein is very similar to that of the D. desulfuricans enzyme and, with the exception of the second S = 1/2 spin system, to that of the prismane protein from wild-type D. vulgaris. Contrary to claims for the D. desulfuricans protein, it is shown here that all data can be fully explained assuming a single [6Fe-6S] cluster, that might be titrated into four different redox states and occurs in up to three different spin systems in the one-electron reduced state.
Stokkermans
J P
JP
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Houba
P H
PH
Pierik
A J
AJ
Hagen
W R
WR
van Dongen
W M
WM
Veeger
C
C
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D016969:Desulfovibrio vulgaris / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C075199:prismane protein, Desulfovibrio vulgaris; D007501:Iron