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Bacterial generation of the anti-greenhouse gas dimethylsulfide: kinetic, spectroscopic, and computational studies of the DMSO reductase system

Polsinelli, Gregory Anthony
http://rave.ohiolink.edu/etdc/view?acc_num=osu1198876055

Abstract Details

2008, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.
The study presented herein provides a better understanding of the global sulfur cycle at the molecular level by exploring the enzymatic process whereby DMS is generated from from dimethylsulfoxide (DMSO) by examining the pathway that leads to the generation of this gas, the DMSO reductase pathway. Resonance Raman (rRaman) spectroscopic studies have also been undertaken in order to determine the roles of two active site residues, W116 and Y114, in the catalytic cycle of substrate turnover in DMSO reductase. We have found that whereas Y114F mutant forms a complex with DMSO substrate and W116F does not form the complex using multiple component analysis (MCA) and rRaman spectroscopy. The reaction mechanism of the properly redox cycled W116F form of the enzyme was determined and is reported. The physiological reductant of DMSO reductase is a pentaheme, membrane-bound c-type cytochrome protein known as DorC. The purification procedure for wild-type DorC has been successfully developed as part of this study. The limiting rate of electron transfer from DorC to DMSO reductase has been determined to be 2.66 s-1 using stopped-flow spectrophotometry and pseudo-first order reaction conditions. This experiment also yielded a Kd of 13.2 μM for binding of DorC to DMSOR. Electron paramagnetic resonance (EPR) spectroscopy has been used to analyze the possible generation of a Mo (V) intermediate and it does not appear as though a Mo (V) state is generated in the course of the reaction. Surface plasmon resonance (SPR or BIAcore) experiments have been undertaken to determine the dissociation constant (Kd) of the complex independent of electron transfer. The Kd was determined to be approximately 30 μM. This study has also produced a computational model of DorC using the computer program Rosetta. From this model, protein docking simulations have been calculated using Hex 4.5 and have produced a compelling working model for the structure of the protein complex with a specific route of electron transfer from the five heme centers of DorC and into the molybdenum center of DMSO reductase identified.
Ross Dalbey (Advisor)
130 p.
Chemistry, Biochemistry
Molybdenum; Dimethylsulfoxide; Dimethylsulfide; Cytochrome

Recommended Citations

Citations

  • Polsinelli, G. A. (2008). Bacterial generation of the anti-greenhouse gas dimethylsulfide: kinetic, spectroscopic, and computational studies of the DMSO reductase system [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1198876055

    APA Style (7th edition)

  • Polsinelli, Gregory. Bacterial generation of the anti-greenhouse gas dimethylsulfide: kinetic, spectroscopic, and computational studies of the DMSO reductase system. 2008. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1198876055.

    MLA Style (8th edition)

  • Polsinelli, Gregory. "Bacterial generation of the anti-greenhouse gas dimethylsulfide: kinetic, spectroscopic, and computational studies of the DMSO reductase system." Doctoral dissertation, Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1198876055

    Chicago Manual of Style (17th edition)

osu1198876055
815
© 2007, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.