Proton-Coupled Structural Changes upon Binding of Carbon Monoxide to Cytochrome cd₁:  A Combined Flash Photolysis and X-ray Crystallography Study^†,‡

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd₁ nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd₁−CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 × 10⁴ and 2.5 × 10³ s^-1 at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400−660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5−8.5). After photodissociation, the CO ligand recombined at a rate of 12 s^-1 (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd₁ in complex with CO was determined to a resolution of 1.57 Å. The structure shows that CO binds to the iron of the d₁ heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR−CO complex indicates changes in the puckering of the d₁ heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d₁ heme. Since the CO ligand binds to heme d₁ and there are structural changes in the d₁ pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d₁ and may regulate the internal electron transfer from heme c to heme d₁.