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

Abstract

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.