Ligand binding and the catalytic reaction of cytochrome caa(3) from the thermophilic bacterium Rhodothermus marinus.

The ligand-binding dynamics and the reaction with O(2) of the fully (five-electron) reduced cytochrome caa(3) from the thermohalophilic bacterium Rhodothermus (R.) marinus were investigated. The enzyme is a proton pump which has all the residues of the proton-transfer pathways found in the mitochondrial-like enzymes conserved, except for one of the key elements of the D-pathway, the helix-VI glutamate [Glu(I-286), R. sphaeroides numbering]. In contrast to what has been suggested previously as general characteristics of thermophilic enzymes, during formation of the R. marinus caa(3)-CO complex, CO binds weakly to Cu(B), and is rapidly (k(Ba) = 450 s(-1)) trapped by irreversible (K(Ba) = 4.5 x 10(3)) binding to heme a(3). Upon reaction of the fully reduced enzyme with O(2), four kinetic phases were resolved during the first 10 ms after initiation of the reaction. On the basis of a comparison to reactions observed with the bovine enzyme, these phases were attributed to the following transitions between intermediates (pH 7.8, 1 mM O(2)): R --> A (tau congruent with 8 micros), A --> P(r) (tau congruent with 35 micros), P(r) --> F (tau congruent with 240 micros), F --> O (tau congruent with 2.5 ms), where the last two phases were associated with proton uptake from the bulk solution. Oxidation of heme c was observed only during the last two reaction steps. The slower transition times as compared to those observed with the bovine enzyme most likely reflect the replacement of Glu(I-286) of the helix-VI motif -XGHPEV- by a tyrosine in the R. marinus enzyme in the motif -YSHPXV-. The presence of an additional, fifth electron in the enzyme was reflected by two additional kinetic phases with time constants of approximately 20 and approximately 720 ms during which the fifth electron reequilibrated within the enzyme.

On the role of the K-proton transfer pathway in cytochrome c oxidase.

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the "peroxy" state, P(r)) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH approximately 7.5. The movement of the Lys is proposed to regulate proton transfer by "shutting off" the protonic connectivity through the K-pathway after initiation of the O(2) reduction chemistry. This "shutoff" prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.

Electron-proton interactions in terminal oxidases.

The cytochrome c and ubiquinol oxidases discussed in this article are membrane-bound redox-driven proton pumps which couple an electron current to a proton current across the membrane. This coupling requires a control of the thermodynamics and/or rates of internal electron- and proton-transfer reactions (termed 'gating'). Therefore, to understand the structure-function relation of these proton pumps, individual electron- and proton-transfer reactions must be investigated. We have undertaken such studies by using a combination of site-directed mutagenesis and spectroscopic techniques. The results show that proton uptake/release upon reduction/oxidation of heme a3 takes place on a ms-time scale through the K-pathway (including Thr(I-359) and Lys(I-362)), but not through the D-pathway (including Asp(I-132) and Glu(I-286)). During reaction of the reduced enzyme with O2, both substrate and pumped protons are taken up through the D-pathway (but not through the K-pathway) in a biphasic process with time constants of 100 microseconds and 1 ms. Thus, the original assignment of the role of the D-pathway (used only for pumped protons) must be revised. Dynamic studies of proton uptake to the enzyme surface show that on the proton-input side, the surface carries a proton-collecting antenna made of carboxylate and histidine residues which enable the enzyme to pick up protons with a rate compatible to the enzyme turnover rate. These results are consistent with the three-dimensional cytochrome c oxidase structure which shows that the entry point to the D-pathway (but not to the K-pathway) is surrounded by a network of histidine residues within a negative electrostatic potential.

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron- and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from approximately 3 microseconds to several milliseconds, reflecting internal electron-transfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV = 1.602 x 10(-19) J), is consistent with a distance of 5-10 A between cytochrome a3 and L. Based on the recently determined high-resolution x-ray structures of bovine and a bacterial cytochrome c oxidase, possible candidates for L and a physiological role for L are discussed.