Determination of the electron self-exchange rate of azurin from Pseudomonas aeruginosa by a combination of fast-flow/rapid-freeze experiments and EPR

Low Reorganization Energy for Electron Self-Exchange by a Formally Copper(III,II) Redox Couple

“…The electron self-exchange rate constant we measured for the LCuOH/[LCuOH] − couple is much larger: k 11 = 3 × 10 4 M –1 s –1 at −88 °C and estimated to be ∼ 9 × 10 6 M –1 s –1 at 25 °C. This latter value is similar to that reported for type 1 Cu proteins (10 5 – 10 6 M –1 s –1 ). ,− Also, the value we measured at −88 °C is 7 orders of magnitude larger than that measured at −90 °C in CH 2 Cl 2 for [Cu II (TMGqu) 2 ] 2+ /[Cu I (TMGqu) 2 ] + self-exchange (10 –3 M –1 s –1 ) (Figure ), which has been touted as a definitive example of a complex held in an entatic state (in a geometry poised between the extremes typical for Cu I and Cu II species and thus expected to undergo fast ET reactions). − Finally, the estimated k 11 value for LCuOH/[LCuOH] − ET at 25 °C (in CH 2 Cl 2 ) is ∼10 or 100 times larger than those for the known Cu III,II couples supported by imine–oxime or tetramide (H –2 Aib 3 ) ligands (in H 2 O), respectively (Figure ). , In separate work, it was shown for the latter system that the Cu III congener is strictly four-coordinate but the Cu II complex is axially coordinated by one or two solvent water molecules .…”

Electron Transfer by Copper Centers

“…[188][189][190][191][192][193][194][195][196][197][198] As noted above, the largest self-exchange rate constants for these copper proteins are in the range of 10 5 -10 6 M -1 s -1 . This is equivalent to the largest values determined for inorganic copper complexes in Table 12 and is also in the same range as the rate constants calculated for the electron-transfer process between stable Cu II L and Cu I L complexes with their corresponding metastable intermediate species after correction for the conformational changes (Table 8).…”

Electron self-exchange in azurin: calculation of the superexchange electron tunneling rate.

“…4 and find kEx 2 x 104 M-1-s-1. This is =50-fold less than that found experimentally at 298 K: 0.4-1.4 x 106 M-1-s-1 (14)(15)(16). This discrepancy, however, is not unacceptably large.…”

“…The self-exchange ET rate for Pseudomonas azurin has been determined by both 'H NMR and rapid-freeze ESR, and the rate constant at 298 K is in the range 0.4-1.4 x 106 M-ls-1 (14)(15)(16)(17). The rate is fairly insensitive to pH and ionic strength and the activation entropy is relatively favorable (15).…”

“…The three-dimensional structure is now available for a number of both natural (wild type) and single-site mutated azurins (9)(10)(11)(12), which makes this group of proteins highly interesting for studies of the different factors that control the rate of long-range ET (13). The rate of self-exchange reactions in both Pseudomonas aeruginosa azurin (14)(15)(16) and Alcaligenes denitrificans azurin (17) have been determined by 'H NMR line broadening and by rapid-freeze ESR as a function of temperature, pH, and ionic strength. The structure ofthe binary complex in the self-exchange reaction is unknown, but the NMR studies described above as well as mutational studies implicate the hydrophobic surface patch surrounding the Cu ligand His-117 (12).…”