Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp. strain J and the structure of novel Az-iso2

Abstract

Two azurin-type blue copper proteins, which are related to the electron-transfer processes involving methylamine/methanol oxidation, have been spectroscopically and electrochemically characterized. The obligate methylotroph Methylomonas sp. strain J gives rise to two azurins (Az-isol and Az-iso2) with methylamine dehydrogenase (MADH-Mj). The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620-630 nm in those of usual azurins. The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character. The half-wave potentials (E1/2) of the two azurins and the intermolecular electron-transfer rate constants (kET) from MADH-Mj to each azurin were determined by cyclic voltammetry. The E1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively. The kET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5. However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1. The structure of Az-iso2 has been determined and refined against 1.6 A X-ray diffraction data. The whole structure of Az-iso2 is quite similar to those of azurins reported already. The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different. These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH.

Structure-based Engineering of Alcaligenes xylosoxidans Copper-containing Nitrite Reductase Enhances Intermolecular Electron Transfer Reaction with Pseudoazurin

“…The catalytic current heights reflect the reac- where C PAZ and C NIR are the solution concentrations of PAZ and CuNIR, respectively, D PAZ is the diffusion coefficient of PAZ, n is the number of electrons, F is the Faraday constant, and A is the area of the working electrode; k is the homogeneous rate constant for the reaction of reduced PAZ with oxidized CuNIR, being equal to the k ET from PAZ to CuNIR. This expression predicts a scan rate-independent, steady-state current that is proportional to the PAZ concentration and the square root of the concentration of CuNIR (30,31). The k ET values calculated with Equation 1 are shown in Table II.…”

Two azurins with unusual redox and spectroscopic properties isolated from the Pseudomonas chlororaphis strains DSM 50083T and DSM 50135

“…The extinction coefficients determined for both proteins are e 626 ¼ 3:40 mM À1 cm À1 and e 280 ¼ 11:1 mM À1 cm À1 , for Az626 and e 624 ¼ 3:34 mM À1 cm À1 and e 276 ¼ 9:53 mM À1 cm À1 , for Az624. The spectrum of the oxidized form of Az626 also exhibits a small peak at 409 nm, probably due to a cytochrome [39]; c [28]; d [40]; e [41]; f [42]; g [43]; h [44]; i [45]; j calculated from ESEEM data reported by Coremans et al [32]; k [46]; l [18]; m Pinho et al, manuscript in preparation; n MADH: methylamine dehydrogenase; o calculated from the aminoacid sequence; p [48]; q [3]; ni, not indicated; nd, not determined.…”

The Significance of the Flexible Loop in the Azurin (Az-iso2) from the Obligate Methylotroph Methylomonas sp. Strain J

“…Therefore, Az-iso2 exhibits a visible absorption and EPR spectra distinct from the typical spectra of azurins. 23 Comparison of the structures between Az-Ad and Az-iso2 in form I and form II…”

“…Moreover, a unique spectroscopic character was observed in the visible absorption and EPR spectra of Az-iso2, and the intermolecular electron-transfer rate constant from MADH to Az-iso2 is larger than that of Az-iso1 by a factor of 5. 23 Up to now, several crystal structures of azurins have been reported. They are the holo, apo, or metal-substituted forms of native and mutant Pseudomonas aeruginosa, 24,25 Alcaligenes denitrificans, 26 -28 Pseudomonas denitrificans, 29 Alcaligenes xylosoxidans, 30,31 and Pseudomonas putida 32 azurins.…”

“…The structure of Az-iso2 in form I refined at 1.6 Å resolution was reported. 23 Here, we performed the structural analysis of form II of Az-iso2, and the structural comparison between form I and form II, and report the structure-function relationship between the structural flexibility around the copper site and its higher electron-acceptor activity with respect to Az-iso1.…”