Overproduction of prismane protein in Desulfovibrio vulgaris (Hildenborough): evidence for a second S= 1/2‐spin system in the one‐electron reduced state

Abstract

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 . desuljiuricans (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.The prismane protein from Desulfovibrio vulgaris (Hildenborough) was the first example of a protein in which a Fe-S cluster with S = 9/2 paramagnetism was discovered [l -31. Since then, high-spin ('superspin') Fe-S clusters with spin systems S > 512 have been described in a number of enzymes: dissimilatory sulfite reductase from D . vulgaris (Hildenborough) [4], CO dehydrogenase from Methanothrix soehngenii [5, 61 and the nitrogenase MoFe protein (the Pclusters in this protein) from Azotobacter vinelandii [7, 81. However, these latter enzymes all contain large numbers of iron atoms (20-30), arranged in multiple, different, clusters. This makes a study of the redox properties and the spectroscopic analysis of the high-spin clusters in these proteins rather complicated. The prismane protcin, on the other hand, was shown to contain only six irons and six acid-labile sulfide ions! molecule [9]. Therefore, this protein was used as a relatively simple model protein for the study of these high-spin Fe-S clusters, although its function is as yet unknown [lo] In the 5+ state, the cluster occurs in two magnetic forms: approximately 90% of the clusters occurs in a high-spin S = 9/2 system and 10% in a S = 1/2 spin system. In the 3 + state, the cluster occurs in a S = 1/2 spin system; the EPR spectrum of...

The hybrid‐cluster protein (‘prismane protein’) from Escherichia coli

“…Midpoint potentials calculated for the redox transitions of the hybrid cluster 2 are −50, +85 and +385 mV. The overall pattern for the three redox transitions of the hybrid cluster is similar to that found for Desulfovibrio HCPs [4,5,8]; midpoint potentials for the ‘+3/+4’ and ‘+4/+5’ transitions in E. coli HCP are, however, ≈ 100 mV more positive than in HCPs from Desulfovibrio species.…”

“…HCP and the gene encoding this protein have previously been isolated from D. vulgaris (Hildenborough) and D. desulfuricans ATCC 27774 [1–4,8,21,22]. Cross‐reactivity with antibodies and hybridization with nucleic acid probes indicated that proteins and DNA sequences with similarity to D. vulgaris HCP and the hcp gene, respectively, also occur in other strictly anaerobic sulfate‐reducing bacteria [2,22].…”

“…Reduction of HCP with HCR and NADH was monitored by following the decrease in absorbance in the 400–500 nm region of the UV/visible absorption spectrum of HCP [2,8]. No reduction of HCP (16 µ m in anaerobic 100 m m Hepes/NaOH, pH 7.5) could be measured over a 30‐min period with either HCR (0.7 µ m ) or NADH (200 µ m ) alone added.…”

“…As another unusual feature for a 4 Fe‐cluster, the hybrid cluster may attain four different oxidation states within the biologically relevant range of redox potentials. Midpoint potentials for the redox transitions were observed at ≈ –150, 0 and +300 mV in the Desulfovibrio proteins [5,8,9]. On the basis of the structure of this second cluster, the protein was renamed ‘hybrid‐cluster protein’[6].…”

Transcriptional regulation of a hybrid cluster (prismane) protein

“…Its up-regulation in the presence of nitrite and nitrate suggests that HCP is involved in nitrite or nitrate reduction. Being a redox-active protein, based on the structural and spectroscopic studies [3,10,11], HCP appears to accomplish an as yet unidentified reaction in the nitrogen cycle. To investigate this possibility HCP has been overexpressed from a recombinant plasmid for physiological studies.…”