Search

The dissimilatory sulfite reductase desulfoviridin was purified from the membrane (mSiR) and the soluble fraction (sSiR) of the sulfate-reducing bacterium Desulfovibrio desulfuricans (Essex). Molecular and spectroscopic properties were determined and compared with the properties of the soluble desulfoviridin from Desulfovibrio vulgaris (Hildenborough). The enzymes were isolated as alpha 2 beta 2 gamma n (n = 1-3) multimers with a relative molecular mass of 200 +/- 10 (gel filtration). Both mSiR and sSiR from D. desulfuricans contained 24 +/- 3 Fe and 18 +/- 3 labile sulfide/200 kDa, respectively, and showed identical EPR spectra. Quantification of the high-spin Fe(III) heme resonances at g of approximately 6 indicated that close to 80% of the siroheme moiety in the enzyme from D. desulfuricans was demetallated. D. desulfuricans sulfite reductase showed S = 9/2 EPR signals with the highest apparent g value at g = 17 as reported for SiR from D. vulgaris. Antibodies raised against the alpha, beta and gamma subunit of the D. vulgaris enzyme exhibited cross-reactivity with the subunits of mSiR and sSiR from D. desulfuricans. N-terminal sequences of alpha, beta and gamma subunits of both mSiR and sSiR from D. desulfuricans were identical and showed a high degree of similarity with the sequences of the corresponding subunits obtained from the D. vulgaris enzyme. During gel filtration of sSiR from D. desulfuricans, under non-denaturing conditions, a small protein (molecular mass approximately 11 kDa) was separated. This 11-kDa protein exhibited cross-reactivity with the antibody raised against the gamma subunit of D. vulgaris sulfite reductase. In the case of D. desulfuricans sulfite reductase, the 11-kDa gamma subunit seems not to be an integral part of the protein and can be obtained from the soluble fraction and during purification of the soluble enzyme.

The active site of Escherichia coli NADPH-sulfite reductase has previously been modeled as a siroheme with its iron bridged to a nearby iron-sulfur cubane, resulting in antiferromagnetic exchange coupling between all iron atoms. The model has been suggested to hold also for other sulfite reductases and nitrite reductases. We have recently challenged the generality of the model with the finding that the EPR of Fe/S in dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris indicates that an S = 9/2 system is not subject to coupling. Siroheme in desulfoviridin is to a large extent demetalated, and therefore coupling is physically impossible. We have now studied examples from a second class of dissimilatory sulfite reductases, desulforubidins, which have their siroporphyrins fully metalated. Desulforubidin from Desulfosarcina variabilis is a 208-kDa alpha 2 beta 2 gamma 2 hexamer. The alpha- and beta-subunits are immunologically active with antibodies raised against the corresponding subunits from D. vulgaris desulfoviridin, whereas the gamma-subunit is not. The desulforubidin contains two fully metalated sirohemes and a total of approximately 15 Fe and approximately 19 S2-. Quantification of high-spin plus low-spin heme EPR signals accounts for all sirohydrochlorin. The frequency-independent (9-35 GHz) effective perpendicular g-values of the high-spin S = 5/2 siroheme (6.33, 5.19) point to quantum mixing with an excited (approximately 770 cm-1) S = 3/2 multiplet. Similar anomalous g-values are observed with sulfite reductases from Desulfovibrio baarsii and Desulfotomaculum acetoxidans. The D. variabilis enzyme exhibits very approximately stoichiometric S = 9/2 EPR (g = 16).(ABSTRACT TRUNCATED AT 250 WORDS)