Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd(1): A combined flash photolysis and X-ray crystallography study

We have investigated dynamic events after flash photolysis of CO from reduc ed cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (f ormerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrom e cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attribut ed to dissociation of CO, was observed. This was followed by a biphasic rea rrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8 .0. Both parts of the biphasic rearrangement phases displayed the same kine tic 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 ac tive 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 relea se. The crystal structure of reduced cytochrome cd(1) in complex with CO wa s determined to a resolution of 1.57 Angstrom. The structure shows that CO binds to the iron of the dr heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the red uced, unligated NiR and the NiR-CO complex indicates changes in the puckeri ng of the d(1) heme as well as rearrangements in the hydrogen-bonding netwo rk and solvent organization in the substrate binding pocket at the dr heme. Since the CO ligand binds to heme dr and there are structural changes in t he dr pocket upon CO binding, it is likely that the proton uptake or releas e observed after flash-induced CO dissociation is due to changes of the pro tonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox pote ntial of heme d(1) and may regulate the internal electron transfer from hem e c to heme d(1).