Electron Pushing: Variants That Alter the Catalytic Distribution of Electrons in Dihydroorotate Dehydrogenase 1B from...

Dihydroorotate dehydrogenase 1B (DHOD1B) is one of several flavoproteins that utilize active half-sites. These enzymes have two flavin cofactors (FAD and FMN) that each interact with a specific reductant/oxidant substrate/product. Electrons gained at one-half-site must be transmitted to the other half-site and iron-sulfur centers between the flavin cofactors serve in this role. DHOD1B from Lactococcus lactis (LlDHOD1B) is a heterodimeric protein that has been shown to fractionally accumulate a flavin bisemiquinone state comprised of equimolar anionic and neutral forms, demonstrating an internal electron distribution equilibrium. Variant forms of LlDHOD1B were designed to perturb predicted or claimed pathways for internal transmission of electrons. Lysine 48 of the PyrD subunit is positioned near the FMN isoalloxazine N5 and the orotate C6-carboxylate. The K48M variant revealed that the anionic semiquinone resides at the FMN cofactor and that the lysine's role is electrostatic, influencing both the pK_a and reduction potential of the FMN. Glutamate 221 of the PyrK subunit stacks with the FAD isoalloxazine. The E221Q variant established that this charge influences the rate of hydride transfer from NADH and the rate of reduction of orotate and accumulates little of the flavin bisemiquinone observed with the WT enzyme. Cysteine 135 of the PyrD subunit serves as the active half-site acid/base. The C135A variant prevented reduction of orotate, permitting the influence of orotate binding on the reduction potential of the FMN cofactor to be determined indicating a +70 mV change in the FMN reduction potential with the association of orotate.