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)O2 complex formation and O2-forced movements affecting hydride transfer to the FAD cofactor and electron-transfer to the Fe(III)O2 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)O2 complex and O2-forced movements. Deoxygenation of the enzyme causes Stark effects on the ferric heme Soret and charge-transfer bands revealing the Fe(III)O2 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 O2 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 fla-vohemoglobin and myoglobin suggesting novel allosteric effector roles for glucose or glucose-6-phosphate in the NO dioxygenase and O2 storage functions. The results support the proposed roles of a ferric O2 intermediate and protein motions in regulating electron-transfer during NO dioxygenase turnover.

Automated summary

Kinetic and structural studies of flavohemoglobin-type NO dioxygenase have revealed the importance of Fe(III)O2 complex formation and O2-induced movements. These investigations provide a spectroscopic method, based on Stark effect theory, for studying the proposed Fe(III)O2 complex and O2-induced movements. Deoxygenation of the enzyme leads to Stark effects on the ferric heme and FAD, revealing the Fe(III)O2 complex and triggering changes in hydride transfer and electron-transfer. Glucose also affects the enzyme's activity by inducing an off state. Amino acid substitutions influence the O2-induced Stark effects on heme spin states and FAD, supporting their proposed roles in the enzymatic mechanism. A conserved glucose or glucose-6-phosphate binding site is identified in the enzyme, suggesting novel allosteric effector roles for glucose in NO dioxygenase function.