Cytochrome a3 hemepocket relaxation subsequent to carbon monoxide photolysis from fully reduced cytochrome ba3 oxidase of Paracoccus denitrificans

Abstract

Time-resolved resonance Raman spectroscopy has been used to probe the structural dynamics at the heme a3 proximal/distal sites subsequent to carbon monoxide photolysis from fully reduced ba3 quinol oxidase from P. denitrificans. The resonance Raman spectra of the fully reduced cyanide-bound ba3, indicate that the 193 cm-l and 208 cm-l modes observed in the fully reduced enzyme disappear and no new lines appear, consistent with the behavior found for the Fe-His stretching mode in other heme proteins and model compounds. Our results indicate that the 193 cm-l and 208 cm-1 modes observed in the equilibrium reduced heme a3 can be assigned to the Fe2+-His of heme a3, in two different conformers of the enzyme. The spectra of the transient species exhibit structural differences relative to the equilibrium geometry of heme a3 (Fig. 1). The most significant of these is a shift of 5 cm-1 to higher frequency of the 208 cm-1 mode in the transient species. Possible explanations for the atypical Fe-His bonding in heme a3 in both the equilibrium and photoproduct states will be discussed. The behavior of the Fe2+-His modes in the photolytic transients of cytochrome ba3 indicate that the 193 cm-1 mode is fully relaxed at 10 ns and at times ∼30 μs subsequent to CO photolysis the proximal heme a3 geometry is fully relaxed. The rate of relaxation of heme a3 is not similar to that observed in the heme a3 transients of cytochrome aa3 oxidase. At later times (td > 100 μs) the appearance of the 213 cm-1 peak signals the onset of CO rebinding to the previously photolyzed heme a3. The Fe-CO stretching mode which is located at 517 cm-1, along with the v(Fe2+-His), places cytochrome ba3 on the Fe2+-His stretching frequency versus Fe-CO stretching inverse correlation curve, characteristic of proteins containing a proximal histidine, including all members of the terminal oxidases. Analysis of the line width of v(Fe-His) during heme a3 relaxation subsequent to CO photolysis reveals the heme a3 pocket evolves in a continuous manner, rather than through a two-state step-function-type mechanism. Collectively, the transient intermediates of heme a3 suggest significant alterations in the nature of the heme-protein dynamics between cytochrome ca3 oxidase and quinol cytochrome ba3 oxidase resulting from specific structural differences within their respective proximal and distal hemepockets.