Cytochrome bd from Azotobacter vinelandii: evidence for high-affinity oxygen binding.
Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements of O2 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give Kd(O2) = 0.5 microM, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 microM); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with O2 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate of O2 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.
Belevich
Ilya
I
Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), 00014, Helsinki, Finland.
Borisov
Vitaliy B
VB
Bloch
Dmitry A
DA
Konstantinov
Alexander A
AA
Verkhovsky
Michael I
MI
D016948:Azotobacter vinelandii / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism; D017937:Cytochrome d Group / Q000737:chemistry / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding