Kinetic and biochemical analysis of electron transfer in dissimilatory metal reduction by Shewanella oneidensis MR-1.

摘要

Biochemical studies were used to further elucidate the pathway of electron transfer across the outer membrane (OM) of Shewanella oneidensis MR-1 and scaling kinetics were conducted to determine the dependence of two OM proteins, OmcA and MtrC, in dissimilatory metal reduction (DMR).;An outer membrane (OM) protein complex MtrC/A/B was purified from anaerobically grown Shewanella oneidensis MR-1. Analytical ultracentrifugation was used to characterize the complex and its molecular mass was determined to be approximately 198 kDa, which is consistent with a 1:1:1 stoichiometry of the individual subunits. This protein complex reduced both soluble and insoluble iron and manganese forms, with rates of reduction correlating to the metal species; i.e. soluble iron was reduced fastest while the most crystalline iron mineral goethite was reduced slowest. This trend is dependent upon the mineral reactivity and not enzyme specificity and therefore these proteins merely act as grounding wires to transfer electrons out of the cell.;Rates of iron reduction were determined at three scales: transient state with purified enzymes, steady state with total membrane (TM) fractions, and steady state with whole cells (WC). Transient state soluble iron reduction kinetic analysis was performed using stop flow techniques to determine molecular rate constants. The reactivity of soluble iron forms for both OmcA and MtrC follows the decrease in the association constant for ligand-metal complexation: EDTA-Fe3+, NTA-Fe3+, and Citrate-Fe3+ . Western blot analysis was used to determine the molar amounts of OmcA and MtrC in TM and WC samples in order to convert specific activity (moles Fe2+ formed/min/mg protein) to a velocity (s-1 : moles Fe2+ formed/sec/moles OmcA). Comparison of rates between transient state and steady state revealed that OmcA and MtrC are kinetically competent to account for whole cell catalysis of soluble iron. When rates of reduction of solid Fe-oxides (goethite) were compared at each kinetic scale, transient-state rate constants were 100 to 1000 times slower than steady-state rate constants. When flavins were included in the reaction mechanism, kinetic competence was exhibited. Thus, electron shuttles, such as flavins, are necessary to account for catalysis of goethite in whole cells.;As observed for iron oxides, OmcA and MtrC were not kinetically competent to account for physiological manganese oxide reduction via direct contact because the relevant rate constants were 3 orders of magnitude too slow. In the presence of flavins, the reaction rates were greatly increased and able to account for the reduction of insoluble manganese oxides in vivo. The extent of flavin stimulation was dependent upon the mineral reactivity, with regards to mineral composition (Fe versus Mn) and mineral structure.