Cytochrome o3 hemepocket relaxation subsequent to carbon monoxide photolysis from fully reduced and mixed valence cytochrome bo3 oxidase

Abstract

SYNOPSISTime-resolved resonance Raman spectroscopy has been used to probe the structural dynamics at the heme 03 proximal and distal sites subsequent to carbon monoxide photolysis from fully reduced and CO mixed-valence cytochrome b03 ubiquinol oxidase. The spectra of the transient species exhibit structural differences relative to the equilibrium geometry of heme 0 3 . The most significant of these is a shift of 4 cm-' to higher frequency of the 208-cm-l mode in the transient species. Our results indicate that the 208-cm-' mode observed in the equilibrium-reduced heme 0 3 , which was recently assigned to the Fe2+-His of heme 0 3 , is located a t 212 cm-' in the 10-ns spectrum. The behavior of the Fez+-His mode in the photolytic transients of cytochrome b03 indicates that at times -10 p s subsequent to CO photolysis the proximal heme 03 geometry is fully relaxed. The rate of relaxation of heme o3 is similar to that observed in the heme a3 transients of cytochrome aa3 oxidase. At later times ( td > 100 p s ) the appearance of the 212-cm-' peak signals the onset of CO rebinding to the previously photolyzed heme 03. Neither the fully reduced nor the mixed-valence species exhibits geminate ligand recombination on a 10-ns time scale. Both species, however, display relaxation of v( Fe-His) to its equilibrium position, a t 208 cm-', on a 10-ps time scale, and ligand rebinding on a 200-ps time scale. Our results indicate that the rate of relaxation of heme 03 and the CO rebinding to heme 03 are independent of the redox state of the low-spin heme b . Collectively, the transient intermediates of heme 03 suggest significant alterations in the nature of the heme-protein dynamics between cytochrome c aa3 oxidase and quinol cytochrome b03 oxidase resulting from specific structural differences within their respective proximal and distal hemepockets.

Observation of the Equilibrium CuB-CO Complex and Functional Implications of the Transient Hemea3 Propionates in Cytochromeba3-CO from Thermus thermophilus

“…where k 1 and k Ϫ1 represent the reversible binding of CO to Cu B , and k 2 is the first-order transfer of CO from Cu B to the heme-Fe (12)(13)(14)(15)(16)(17)(18)(19)(20). The thermal dissociation rate of the heme-CO complex of bovine fully reduced cytochrome c oxidase-CO is very slow (0.023 s Ϫ1 ) and thus, k Ϫ2 can be neglected (13).…”

Oxygen-linked Equilibrium CuB-CO Species in Cytochrome ba3 Oxidase from Thermus thermophilus

“…Because of the unusual ligand-binding and kinetic properties of the binuclear center, cytochrome ba 3 oxidase is unique among the heme-copper oxidases in that it is susceptible to a detailed kinetic analysis of its ligand dynamics (4,7). The binding of CO to the binuclear center of ba 3 follows that found in all heme-copper oxidases and proceeds according to the Scheme 1 (7)(8)(9)(10)(11)(12).…”