Sulfur Biogeochemistry—Past and Present

6 V. Brüchertnot yet been resolved. Assimilatory sulfite reductases, althoughdifferent in structure, appear to catalyze the reduction of sulfitein a single 6-electron transfer, without the formation of sulfurintermediates (Crane et al., 1995).Isotope Fractionation ModelsThe early experimental results by Harrison and Thode(1958), Kaplan and Rittenberg (1964), and Kemp and Thode(1968) were used by Rees (1973) to construct a flow model forsulfate transport through the cell during bacterial sulfate reduction.Isotope fractionation can be expressed (a) during cellularuptake, (b) during reduction to sulfite, and (c) during reductionto sulfide (Fig. 2). Rees (1973) summarized the combined effectsfor isotope fractionations and introduced the concept of ratelimitingsteps. According to this concept, isotope fractionationsare not produced downstream from the rate-limiting step. If therate-limiting step is the cellular uptake of sulfate across the cellmembrane, the largest possible isotope fractionation is that associatedwith cellular uptake. Conversely, if the rate-limiting stepoccurs downstream in the sulfate reduction process (e.g., at thedissimilatory sulfite reductase), isotope fractionations can be producedat all steps upstream from and at the rate-limiting step. Ingeneral, it can be expected that the bacterial cell will tune the cellularuptake and the energy-consuming activation of sulfate to therate of electron donor uptake. Consequently, the rate of sulfateand intermediate sulfur compound turnover depends on the rateat which the electron-transport chain operates. Furthermore, therate of the electron-transport chain influences the electrochemicalpotential, which in turn regulates the rate of sulfate transportacross the cell membrane.These considerations are the basis for kinetic models ofisotope fractionation during bacterial sulfate reduction. Generally,the overall isotope fractionation is regulated by two factors:(1) the rate of cellular uptake of sulfate relative to the sulfatedemand by the enzymes catalyzing the reduction of APS andsulfite, and (2) the isotope fractionation of the reduction process.The overall isotope fractionation epsilon (ε BSR) is the sum ofthese two effects and can be expressed byε BSR= f • ε transport+ (1− f) • ε reduction, (1)where 0 < f < 1. The implication of the Rees (1973) model is thatisotope discrimination of sulfate occurs inside the cell and thatthe isotope composition of the sulfate transported into the cellis that of the ambient environment. Inside the cell, the specificenzymatic kinetics of the 34 S-APS and 32 S-APS molecule at theAPSR and of the 34 S-sulfite and 32 S-sulfite at the DSR regulatethe isotope composition of the sulfur compounds. The selectiveconsumption of the light isotope would leave the residual pool ofthe intermediate sulfur compounds, i.e., internal sulfate, APS, orsulfite, enriched in 34 S. To preserve mass balance, at steady-state34S-enriched intermediate sulfur compounds or sulfate must continuouslyleave the cell.There are energetic considerations that would support analternative model for the cellular regulation of isotope fractionation.As indicated above in the section “Sulfate Transport andEnzymatic Reduction: Energetic Considerations,” it is unlikelythat a bacterial cell will only reduce part of the sulfate transportedat the expense of ATP into the cell and then transportunused 34 S-enriched sulfate back out of the cell. Furthermore,it is energetically costly for the cell to lose sulfate from insidethe cell back to the environment because reversed transport ofa negatively charged species such as sulfate to the outside ofthe cell lowers the membrane potential. Reversed transport(i.e., leakage of 34 S-enriched sulfate from the cell) negativelyaffects the fundamental requirement for the maintenance of cellmetabolism, because the cell de-energizes itself (Cypionka,1995; White, 1995). Therefore, an alternative fractionationmodel would be to consider a sulfur isotope gradient across thecell membrane due to the fractionation created by the specificactivity of the APSR and DSR. The specific activity of the twoenzymes, in turn, is regulated by the rate of the electron-transportchain (Fig. 2). In this case, sulfate transported across thecell membrane is already fractionated (i.e., 32 S-enriched relativeto the ambient environment) exactly to the extent that the twoenzymes APSR and DSR fractionate APS and intermediate sulfurcompounds. Equation 1 is also valid for this modification. Ifthe rate of transport across the cell membrane controls the rateof sulfate reduction, then uptake is the rate-limiting step for sulfatereduction. In this case, sulfate availability in the micrometer-scaleambient environment around the cell is limited, withthe consequence that the diffusing sulfate is not fractionatedrelative to sulfate in the ambient environment. Alternatively, ifthe rate of reduction is the rate-limiting step, then the enzymescan produce isotope fractionation. This is because sulfateavailability in the ambient environment is unlimited, and thediffusing sulfate can be fractionated. Implicit to the model isthat there are no intermediates and that that there is no reversetransport. This hypothesis should be tested with appropriateexperiments.ENZYME-SPECIFIC FRACTIONATION OF THEADENOSINE PHOSPHOSULFATE REDUCTASE ANDTHE DISSIMILATORY SULFITE REDUCTASEAlthough these data are central in our understanding of sulfurisotope fractionation, there is only one study that determinedthe isotope fractionation of the enzyme DSR, and in this study,only cell-free extracts were used (i.e., the enzyme was not purified).Kemp and Thode (1968) reported an isotope fractionationof 18‰ for the dissimilatory sulfite reductase of Desulfovibriodesulfuricans. At present, comparable information is not availablefor the adenosine phosphosulfate reductase (APSR). Rees(1973) derived an isotope fractionation of 25‰ for the APSRby difference after subtracting an isotope fractionation of 25‰for the DSR from a total isotope fractionation of 47‰ measuredexperimentally (Kaplan and Rittenberg, 1964).