Pulse Radiolysis Studies of Temperature Dependent Electron Transfers among Redox Centers inba₃-CytochromecOxidase fromThermus thermophilus: Comparison of A- and B-Type Enzymes

Abstract

The functioning of cytochrome c oxidases involves orchestration of long-range electron transfer (ET) events among the four redox active metal centers. We report the temperature dependence of electron transfer from the Cu(A)(r) site to the low-spin heme-(a)b(o) site, i.e., Cu(A)(r) + heme-a(b)(o) → Cu(A)(o) + heme-a(b)(r) in three structurally characterized enzymes: A-type aa(3) from Paracoccus denitrificans (PDB code 3HB3 ) and bovine heart tissue (PDB code 2ZXW ), and the B-type ba(3) from T. thermophilus (PDB codes 1EHK and 1XME ). k,T data sets were obtained with the use of pulse radiolysis as described previously. Semiclassical Marcus theory revealed that λ varies from 0.74 to 1.1 eV, H(ab), varies from ∼2 × 10(-5) eV (0.16 cm(-1)) to ∼24 × 10(-5) eV (1.9 cm(-1)), and βD varies from 9.3 to 13.9. These parameters are consistent with diabatic electron tunneling. The II-Asp111Asn Cu(A) mutation in cytochrome ba(3) had no effect on the rate of this reaction whereas the II-Met160Leu Cu(A)-mutation was slower by an amount corresponding to a decreased driving force of ∼0.06 eV. The structures support the presence of a common, electron-conducting "wire" between Cu(A) and heme-a(b). The transfer of an electron from the low-spin heme to the high-spin heme, i.e., heme-a(b)(r) + heme-a(3)(o) → heme-a(b)(o) + heme-a(3)(r), was not observed with the A-type enzymes in our experiments but was observed with the Thermus ba(3); its Marcus parameters are λ = 1.5 eV, H(ab) = 26.6 × 10(-5) eV (2.14 cm(-1)), and βD = 9.35, consistent also with diabatic electron tunneling between the two hemes. The II-Glu15Ala mutation of the K-channel structure, ∼24 Å between its CA and Fe-a(3), was found to completely block heme-b(r) to heme-a(3)(o) electron transfer. A structural mechanism is suggested to explain these observations.

The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping

“…1B) from the observed firstorder rate constants for oxidation of heme b 560 at different temperatures, as well as from the data published on the oxidation rate of heme b 560 in pulse radiolysis experiments [16]. Although the observed heme b 560 oxidation rates in the stopped-flow experiments are approximately twofold lower than the rates determined at the same temperature in the pulse radiolysis experiments, our data (E a~3 6 kJ/mol; Fig.…”

“…The internal equilibrium between heme b 560 and Cu A is given by forward (k 2 ) and reverse (k −2 ) rate constants of 4621 s −1 and 3466 s −1 indicating a 7.5 (±5.8) mV higher midpoint potential for heme b 560 compared to Cu A , contrast to earlier pulse radiolysis studies [16], where a 16 mV lower midpoint potential for heme b 560 was reported. However, the sum of k −2 and k 2 (8087 s −1 ), which is k obs 2 is nearly equal to the values of 8380 s −1 and 8500 s −1 for 5°C and 10°C, respectively, determined in [16].…”

“…The oxidation/reduction rates that we observe for heme b 560 in stopped-flow experiments are approximately a factor of 2 lower than observed in pulse radiolysis experiments while yielding a similar activation energy [9,16]. For the freeze experiments (Fig.…”

“…4) we calculate a similar k obs 2 (~8000/s) as in [16] but the individual values of k −2 and k 2 yield a 7.5 mV higher midpoint potential of heme b 560 compared to Cu A whereas a value of 16 mV lower was previously observed [16]. Although these differences might be due to different experimental techniques, the crucial difference is that the stopped-flow and freezequench experiments reported here were performed starting with the reduced enzyme in reaction with O 2 , thus monitoring the oxidative half-cycle of the reaction, whereas the pulse radiolysis studies were performed in the absence of oxygen, monitoring injection of a single electron, the initial step of the reductive half-cycle [16]. It thus appears that the energy barrier to overcome for ET between heme b 560 and heme a 3 is approximately the same in both half-cycles and is not affected by the reduction and/or ligand state of the binuclear center or that of other redox centers.…”

“…The basis for this difference has been proposed to be due to the different timing of electron and proton transfer events between the two enzymes [10], or because of the lowered proton uptake activity of the proton pathway in cytochrome ba 3 oxidase [12][13][14] or to differences in the intramolecular electron transfer vs. Type A CcOs [10,[15][16][17]. A proton gating mechanism is required to prevent the leaking back of protons towards the negative side of the membrane.…”