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.
Ross
Daniel E
DE
Center for Environmental Kinetics Analysis, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Brantley
Susan L
SL
Tien
Ming
M
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology*; D013379:Substrate Specificity
D001425:Bacterial Outer Membrane Proteins; D003574:Cytochrome c Group; D007501:Iron; C433527:MtrC protein, Shewanella; D010088:Oxidoreductases