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

Abstract

1؉ -CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu B between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

“…In particular, binding of CO to this enzyme leads to formation of only 70% of the heme a 3 -CO complex (21,24), with the remaining 30% forming a CuB-CO complex (25). To estimate the extent of NO binding to heme a 3 , the inset of Figure 1B shows the second derivative of the Soret spectra of the unligated and NO-bound forms.…”

CO impedes superfast O 2 binding in ba 3 cytochrome oxidase from Thermus thermophilus

“…However, crystallographic studies using Xe and Kr to map out the O 2 pathway within ba 3 have indicated that there is no straight-access line from Xe1, the site closest to the binuclear cavity, to Cu B , and that Trp229 and His283 appear to form a barrier that O 2 needs to circumvent to access the dinuclear center (21). These observations, together with the long lifetime of the transient Cu B þ -CO complex in ba 3 (14), suggest that Cu B may not necessarily act as an obligatory stop for O 2 entry to heme a 3 in ba 3 -with-CO. In the absence of CO, the entry of O 2 to heme a 3 in ba 3 may or may not involve prior binding to Cu B .…”

“…The obligatory path of CO to and from heme a 3 in the bovine enzyme has been proposed to involve the binding of CO to Cu B þ (13,25), and an analogous mechanism has been postulated for ba 3 (14,26); the O 2 ligand has been proposed to follow the same path (27). However, crystallographic studies using Xe and Kr to map out the O 2 pathway within ba 3 have indicated that there is no straight-access line from Xe1, the site closest to the binuclear cavity, to Cu B , and that Trp229 and His283 appear to form a barrier that O 2 needs to circumvent to access the dinuclear center (21).…”

“…In ba 3 , the thermal dissociation of CO from heme a 3 2þ in the dark is significantly faster (0.8 s −1 ) (5) than in the bovine aa 3 (0.023 s −1 ) (3,13), and therefore CO flow-flash experiments on ba 3 require fast mixing; such experiments have recently been reported (6,7). Moreover, the Cu B þ -CO complex formed following CO photolysis from heme a 3 2þ in ba 3 decays with a lifetime of approximately 30 ms (14), a rate much slower than that of O 2 binding to heme a 3 2þ in the aa 3 -oxidases (approximately 10 μs at 1 mM O 2 ). This raises the question whether the O 2 binding in ba 3 is compromised by the fate of the photodissociated CO.…”

Direct Detection of Fe(IV)=O Intermediates in the Cytochrome aa3 Oxidase from Paracoccus denitrificans/H2O2 Reaction

“…In the presence of an H-bonded His-411, electron density is pushed into the antibonding * orbitals of Fe(IV)ϭO, weakening the bond, and thus, shifting the Fe(IV)ϭO stretching frequency from 804 to 790 cm Ϫ1 . The details on how the 804 -790 cm Ϫ1 oxoferryl transition is specifically tuned to initiate the H-bonding interaction and whether it is coupled to the heme a 3 -propionatesAsp-H 2 O protonic connectivity, in a similar manner to that found in cytochrome ba 3 from T. thermophilus (69), remain to be determined.…”