Kinetic studies of the reactions of O(2) and NO with reduced Thermus thermophilus ba(3) and bovine aa(3) using photolabile carriers.
The reactions of molecular oxygen (O(2)) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba(3) and bovine heart aa(3) were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O(2) and NO complexes. The binding of O(2)/NO to reduced ba(3) in the absence of CO occurs with a second-order rate constant of 1×10(9)M(-1)s(-1). This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O(2)/NO binding in ba(3) is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O(2) and NO access to the active site in Tt ba(3), and that traditional CO flow-flash experiments do not accurately reflect the O(2) and NO binding kinetics in ba(3). We suggest that in ba(3) the binding of O(2) (NO) to heme a(3)(2+) causes rapid dissociation of CO from Cu(B)(+) through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu(B)(+). These findings indicate that structural differences between Tt ba(3) and the bovine aa(3) enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA. olof@ucsc.edu
Olöf
O
Einarsdóttir
Chie
C
Funatogawa
Tewfik
T
Soulimane
Istvan
I
Szundi
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002417:Cattle; D003573:Cytochrome b Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000528:radiation effects; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D013053:Spectrophotometry / Q000379:methods; D016963:Thermus thermophilus / Q000201:enzymology* / Q000378:metabolism / Q000528:radiation effects
D003573:Cytochrome b Group; D009569:Nitric Oxide; D002248:Carbon Monoxide; C058557:cytochrome ba3; D003576:Electron Transport Complex IV; D010100:Oxygen