The dissimilatory sulfite reductase from Desulfosarcina variabilis is a desulforubidin containing uncoupled metalated sirohemes and S = 9/2 iron-sulfur clusters

Abstract

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)

The Crystal Structure of Desulfovibrio vulgaris Dissimilatory Sulfite Reductase Bound to DsrC Provides Novel Insights into the Mechanism of Sulfate Respiration

“…Cofactor quantification of dSiRs is quite disparate, with studies reporting from 2 to 4 sirohemes and from 10 to 32 non-heme irons per ␣ 2 ␤ 2 module (13,(17)(18)(19)(20). The nature of the catalytic cofactor has also been disputed, with some studies proposing a cubanesiroheme arrangement similar to that found in aSiRs (13,19) and other studies proposing the presence of higher nuclearity high spin iron-sulfur clusters (18,20,21). Finally, a most important and unresolved question is the nature of the physiological electron donor to dSiR.…”

Isolation and characterization of a desulforubidin-containing sulfate-reducing bacterium growing with glycolate

“…However, a sulfite reductase of the desulforubidin-type was detected spectroscopically in the cytoplasmic fraction after separation from interfering cytochromes by chromatography. The absorption spectrum of the purified protein exhibited a maximum at 545 nm, typical of sulfite reductases of the desulforubidin-type (Lee et al 1973;Arendsen et al 1993); in addition, further characteristic peaks were observed at 279, 396, and 580 nm (Fig. 2).…”

“…According to this analysis, the enzyme was 98% pure. The enzyme preparation was highly purified also on the basis of UV/Vis spectroscopy; the purity indices were 3.77 (398 nm/545 nm), and 0.5 (398 nm/280 nm), in comparison to 3.65 (398 nm/545 nm), and 0.35 (398 nm/545 nm) for desulforubidin from Desulfosarcina variabilis (Arendsen et al 1993). Desulfoviridins of two Desulfovibrio vulgaris strains have been shown previously to resolve into two (Seki et al 1979) or three (Wolfe et al 1994) distinct forms during anion-exchange chromatography.…”

“…Dissimilatory sulfite reductases known so far all comprise a heteropolymeric ~2~27zstructure with molecular masses in the range of 150 to 230 kDa and with approximate subunit molecular masses of 50 (~), 40 (~), and 12 kDa (7) (Pierik et al 1992;Hansen 1994); it was only recently that the small subunit was discovered (Pierik et al 1992). The desulforubidin purified previously from Desulfosarcina variabilis has been reported to have subunits of 50 (~), 42.5 (~), and 12 kDa (7) molecular mass (Arendsen et al 1993). As revealed by SDS-PAGE of the purified desulforubidin from strain PerGlyS, we found three subunits of 42.5, 38.5, and 13 kDa corresponding to the c~7-composition of dissimilatory sulfite reductases, but the sizes of the c~ and [3 subunits differ significantly from those of other sulfite reductases.…”

Similarities in the Architecture of the Active Sites of Ni‐Hydrogenases and Fe‐Hydrogenases Detected by Means of Infrared Spectroscopy

“…A number of enzymes were purified by published procedures : Ni-hydrogenase from Alcaligenes eutrophus (soluble NAD+-reducing enzyme; cells were obtained from G. Haverkamp and C. G. Friedrich, Dortmund, Germany;Schneider et al, 1979;Friedrich et al, 1982); Ni-hydrogenase from C. vinosum (strain DSM 185) (Coremans et al, 1992b); Fe-hydrogenase ( Van der Westen et al, 1978) and prismane protein (Stokkermans et al, 1992) from D. vulgaris (Hildenborough); dissimilatory sulfite reductase from Desulfbsarcinu variabilis (Arendsen et al, 1993); ferredoxin from Megadphaera elsclenii (Gillard et al, 1965); and rubredoxin from Pyrococcus furiosus (Blake et al, 1991).…”