D-2-hydroxyglutarate dehydrogenase (Uniprot ID: Q9I6H4, D2HGDH) from Pseudomonas aeruginosa PAO1 is a Zn²+-dependent flavoenzyme that catalyzes the oxidation of D-2-hydroxyglutarate or D-malate to 2-ketoglutarate or oxalacetate. NAD(P)H: quinone reductase PA0660 (Uniprot ID: Q9I5R1, PaNQR) is an FMN-dependent reductase from P. aeruginosa PAO1, which catalyzes the two-electron reduction of quinones to hydroquinones. In this thesis, the mechanistic consequences of substituting the native Zn²⁺ with Ni²⁺ were investigated using steady-state kinetics, rapid kinetics, and kinetic solvent viscosity effects. PaNQR was examined to elucidate how distal charged residues influence cofactor recognition by a double charge-reversal mutation, rapid kinetics, and structure prediction. The results revealed that Zn²⁺ supports a mechanism limited by hydride transfer and product dissociation, whereas Ni²⁺ substitution shifts the rate-limiting process toward an internal isomerization in PaD2HGDH. PaNQO K274E/K277E at the cofactor binding site perturbed the NADH and NADPH preference. Steady-state kinetics, rapid-state kinetics, and structural prediction together demonstrated that electrostatic interactions tuned the cofactor preference in PaNQR. Overall, these studies provide complementary mechanistic insights into metal-dependent catalysis and cofactor utilization in P. aeruginosa flavoenzymes.