Dioxygen and glucose force motion of the electron-transfer switch in the iron(III) flavohemoglobin-type nitric oxide dioxygenase.

Kinetic and structural investigations of the flavohemoglobin-type NO dioxygenase have suggested critical roles for transient Fe(III)O complex formation and O-forced movements affecting hydride transfer to the FAD cofactor and electron-transfer to the Fe(III)O complex. Stark-effect theory together with structural models and dipole and internal electrostatic field determinations provided a semi-quantitative spectroscopic method for investigating the proposed Fe(III)O complex and O-forced movements. Deoxygenation of the enzyme causes Stark effects on the ferric heme Soret and charge-transfer bands revealing the Fe(III)O complex. Deoxygenation also elicits Stark effects on the FAD that expose forces and motions that create a more restricted NADH access to FAD for hydride transfer and switch electron-transfer off. Glucose also forces the enzyme toward an off state. Amino acid substitutions at the B10, E7, E11, G8, D5, and F7 positions influence the Stark effects of O on resting heme spin states and FAD consistent with the proposed roles of the side chains in the enzyme mechanism. Deoxygenation of ferric myoglobin and hemoglobin A also induces Stark effects on the hemes suggesting a common 'oxy-met' state. The ferric myoglobin and hemoglobin heme spectra are also glucose-responsive. A conserved glucose or glucose-6-phosphate binding site is found bridging the BC-corner and G-helix in flavohemoglobin and myoglobin suggesting novel allosteric effector roles for glucose or glucose-6-phosphate in the NO dioxygenase and O storage functions. The results support the proposed roles of a ferric O intermediate and protein motions in regulating electron-transfer during NO dioxygenase turnover.