Kinetic Characterization of OmcA and MtrC,Terminal Reductases Involved in Respiratory Electron Transfer for Dissimilatory Iron Reduction in Shewanella oneidensis MR-1

We have used scaling kinetics and the concept of kinetic competence to elucidate the role of hemeproteins OmcA and MtrC in iron reduction by Shewanella oneidensis MR-1. Second-order rate constants for OmcA and MtrC were determined by single-turnover experiments. For soluble iron species, a stopped-flow apparatus was used, and for the less reactive iron oxide goethite, a conventional spectrophotometer was used to measure rates. Steady-state experiments were performed to obtain molecular rate constants by quantifying the OmcA and MtrC contents of membrane fractions and whole cells by Western blot analysis. For reduction of soluble iron, rates determined from transient-state experiments were able to account for rates obtained from steady-state experiments. However, this was not true with goethite; rate constants determined from transient-state experiments were 100 to 1,000 times slower than those calculated from steady-state experiments with membrane fractions and whole cells. In contrast, addition of flavins to the goethite experiments resulted in rates that were consistent with both transient- and steady-state experiments. Kinetic simulations of steady-state results with kinetic constants obtained from transient-state experiments supported flavin involvement. Therefore, we show for the first time that OmcA and MtrC are kinetically competent to account for catalysis of soluble iron reduction in whole Shewanella cells but are not responsible for electron transfer via direct contact alone with insoluble iron-containing minerals. This work supports the hypothesis that electron shuttles are important participants in the reduction of solid Fe phases by this organism.Microbial processes play a central role in the cycling of organic and inorganic compounds on Earth. A select number of these compounds are assimilated by organisms and used as building material for the cell. In addition, a large number of organic and inorganic compounds are used as electron donors and acceptors for respiratory metabolism. Iron, one of the most abundant elements in the Earth''s crust, is both assimilated by life and utilized as an electron donor (Fe²⁺) and acceptor (Fe³⁺) in respiratory metabolism. Due to the importance of iron, microorganisms directly impact the fate and transport of iron, and these Fe impacts indirectly influence the (bio)geochemical cycles of many other elements, including the carbon cycle (38). Furthermore, respiratory metabolism evolved in microorganisms prior to the emergence of oxygenic photosynthesis and was very versatile in using a large number of both organic and inorganic terminal electron acceptors (TEAs) (29). Therefore, the use of iron as a TEA predates the use of molecular oxygen among organisms on Earth. When metals are used as the TEA, this process is referred to as dissimilatory metal reduction (DMR) or, in the case of iron, dissimilatory iron reduction (DIR). Interest in microbial DMR has intensified upon discovery of the ability of DMR bacteria to catalyze reactions of environmentally relevant contaminants, including radionuclides such as U(VI) (1, 19, 20, 24, 30, 45, 47).DMR has been extensively studied in the facultative anaerobe Shewanella oneidensis MR-1 due to its respiratory versatility (for a review, see references 22, 23, 39, and 49). This bacterium also has the ability to utilize soluble and insoluble forms of iron and manganese as TEAs (12, 17, 25, 29, 33, 36). Genetic and biochemical studies show that reduction of the soluble TEAs occurs at the cytoplasmic membrane (CM), the location of the proton motive force-generating electron transport. However, in utilization of insoluble oxides, CM-localized electrons need to traverse the periplasm and the outer membrane (OM) to reach the extracellular matrix. Three mechanisms of transfer of OM-localized electrons to metal oxides have been hypothesized: (i) electron transfer through direct contact with the metal oxide, (ii) cyclic reduction/oxidation of electron shuttles, and (iii) solubilization of the metals with chelators, followed by diffusion to and across the OM to the CM. For a review of the proposed mechanisms, see reference 15.While there are very few reports of bacteria producing chelators for DIR (51), there are more data to support the other two mechanisms. For example, using atomic force microscopy (AFM), Lower et al. (26) measured binding between both OM hemeproteins OmcA and MtrC and the iron oxide hematite and reported that OmcA shows a greater affinity toward hematite than does MtrC. These workers suggested that this tight binding is a prerequisite for direct electron transfer. Xiong et al. (55) also showed tight binding between OmcA and hematite by using dynamic light scattering and fluorescence correlation spectroscopy. Previous work in our laboratory (43, 44) demonstrating the rates of metal oxide reduction by membrane fractions from Shewanella also supported a direct-contact mechanism. Direct contact between cellular components and iron oxides is also consistent with the nanowire mechanism proposed by Gorby et al. (14).In support of the electron shuttle hypothesis, two laboratories independently showed that S. oneidensis produces extracellular flavins, which may act as electron-shuttling agents (31, 52). The involvement of flavins as an electron shuttle would explain previous reports that Shewanella secretes a small, quinone-like compound involved in electron transfer (41) and can utilize sterically sequestered iron located in alginate beads (18).While the previous studies described above provide compelling evidence for their respective mechanisms, none of the mechanisms have been subjected to detailed kinetic analysis. Kinetic studies, while not able to prove a mechanism, can be used to eliminate proposed mechanisms if rates of a reaction measured with purified enzymes, as determined in vitro, cannot account for the rates observed in whole cells. We previously described the importance of kinetic studies and how to apply these kinetic studies to DIR (6, 43, 44). Briefly, rate constants are determined by in vitro studies with purified enzymes. The kinetics of the enzyme of interest is then studied in the whole cell (or in subcellular fractions). The rate constants are then compared between these systems to determine whether the rate of the reactions catalyzed by the enzyme can account for the rates observed in the cell.In our previous work, we performed kinetic studies on membrane fractions of S. oneidensis (43, 44). In the present study, this kinetic characterization is extended to purified enzymes and with whole cells for the purpose of determining kinetic competence. While our major focus is on the reduction of insoluble iron forms, for comparative purposes and as a control to validate our studies with the insoluble oxides, our study also includes a kinetic analysis of soluble chelated iron forms. We have focused our kinetic studies on two OM-localized hemeproteins, OmcA and MtrC. Previous genetic knockout studies show the importance of these enzymes in metal oxide reduction as potential terminal metal reductases (2, 35, 37). We describe both transient- and steady-state kinetic analyses. While both hemeproteins have been studied by stopped-flow analysis (48, 53) and by steady-state methods in whole cells (4), no study has yet attempted to infer mechanisms from scale-up kinetic studies (where kinetic constants from transient-state experiments are compared with those obtained from steady-state experiments). Our studies were thus performed at three different kinetic scales: (i) transient-state iron reduction using purified OmcA and MtrC, (ii) steady-state kinetics with purified total membrane (TM) fractions, and (iii) steady-state whole-cell kinetics.