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Hydrogenase Inhibition by O2: Density Functional Theory/Molecular Mechanics Investigation

Dogaru, Daniela

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2008, Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, College of Science.

[Fe-Fe]-hydrogenases are enzymes that reversibly catalyze the reduction of protons to molecular hydrogen, which occurs in anaerobic media. In living systems, [Fe-Fe]-hydrogenases shift the reversible reaction towards H2 formation. The [Fe-Fe]-hydrogenase H-cluster is the active site, which contains two iron atoms (Fep-Fed, i.e., proximal and distal iron). Because most experimental and theoretical investigations confirm that the structure of di-iron air inhibited species is FepII-FedII-O-O-H-, O2 has to be prevented from binding to Fed in all di-iron subcluster oxidation states in order to retain a catalytically active enzyme. By understanding the catalytic processes of metalloenzymes, researches are enabled to produce an excellent source of fuel and energy storage (H2) for the future, which is clean and highly energetic when reacted with oxygen.

H-cluster oxidation in gas phase, and in aqueous enzyme phase, has been investigated by means of quantum mechanics (QM) and combined quantum mechanics-molecular mechanics (QM/MM).

The inhibitory process occurs at the coordination site, distal iron (Fed), of the catalytic H-cluster. The processes involved in the H-cluster oxidative pathways are O2 binding, e- transfer, protonation, and H2O removal. We found that oxygen binding is non-spontaneous in gas phase, and spontaneous for aqueous enzyme phase where both Fe atoms have oxidation state II; however, it is spontaneous for the partially oxidized and reduced clusters in both phases. Hence, in the protein environment the O2-inhibited H-cluster is obtained by means of exergonic reaction pathways.

A unifying endeavor has been carried out for the purpose of understanding the thermodynamic results vis-à-vis several other performed electronic structural methods, such as frontier molecular orbitals (FMO), natural bond orbital partial charges (NBO), and H-cluster geometrical analysis.

Since hydrogenases become O2 inactivated, residue mutations were carried out in order to make them O2 resistant. Residue mutations consist of deletions and substitutions 8 Å radially outward from Fed. In order to screen the polar residues (in the 8 Å apoenzyme layer), individual residue deletions were carried out to determine what residue substitutions should be made to improve O2 inhibition. Residue deletions and substitutions were performed for three di-iron subcluster oxidation states, FepII-FedII, FepII-FedI, and FepI-FedI of [Fe-Fe]-hydrogenase. From the screened residues, two deletions (ΔThr152, and ΔSer202) were found most effective in hindering O2 binding to Fed. The two-residue deletions, ΔThr152 and ΔSer202, on FepII-FedII hydrogenase, gave ΔGQM/MM = +5.4 kcal/mol, which evidently hinders O2 binding. An improvement in Gibbs’ energy (+4.4 kcal/mol) has also been found for FepI-FedI hydrogenase. Comparing the simultaneous residue deletions (ΔThr152 and ΔSer202) with the dual residue substitutions (Thr152Ala, and Ser202Ala), a small difference in Gibbs’ energy has been found (ΔGQM/MM ~ +2 kcal/mol), for O2 binding, which is attributed to an overall charge of approximately zero for alanine. The eventual propose hydrogenase mutation in molecular biology laboratory should avail researchers in using it for the full cells of the future.

Valentin Gogonea, Ph.D. (Committee Chair)
Mekki Bayachou, Ph.D. (Committee Member)
Stan Duraj, Ph.D. (Committee Member)
John Turner, Ph.D. (Committee Member)
John Masnovi, Ph.D. (Committee Member)
Keith Kendig, Ph.D. (Committee Member)
109 p.

Recommended Citations

Citations

  • Dogaru, D. (2008). Hydrogenase Inhibition by O2: Density Functional Theory/Molecular Mechanics Investigation [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1231721611

    APA Style (7th edition)

  • Dogaru, Daniela. Hydrogenase Inhibition by O2: Density Functional Theory/Molecular Mechanics Investigation. 2008. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1231721611.

    MLA Style (8th edition)

  • Dogaru, Daniela. "Hydrogenase Inhibition by O2: Density Functional Theory/Molecular Mechanics Investigation." Doctoral dissertation, Cleveland State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1231721611

    Chicago Manual of Style (17th edition)