Azurin difference absorption

Figure 1. Reduced-minus-oxidized difference absorption spectrum of Ps. aeruginosa azurin. Sample and reference cell contained 6.8 X 10 5 M solutions of reduced and oxidized protein, respectively. Hydrogen, with platinum black as catalyst, was used as reductant (1). Medium ...

Relaxation Kinetics. The details of the experimental procedure have been described earlier (14). 0.1 M phosphate buffer, pH 7.0, containing 2 X 10"5 M EDTA was used in all relaxation experiments. These were performed with solutions of different initial reagent composition— either ferrocyanide was added to oxidized azurin or ferricyanide to reduced azurin. Temperature jumps of 2.9° or 4.7° were applied to the reaction solution. The subsequent transmission changes were monitored at 625 nm (absorption of oxidized azurin) or 420 nm (absorption of ferricyanide ). Each plotted value of the relaxation time or amplitude represents the average of at least four measurements. 

The additional effects in the aromatic region of the difference spectrum (250-300 nm) are probably caused by aromatic transitions which are influenced by the redox state of the copper. The shoulder at 270 nm, which occurs in all three proteins, could result from an increase in tyrosine absorption. In this context, it is interesting to recall that Tyr 108 (azurin numbering), which is relatively close to the proposed copper ligands Cys 112 and Met 121, is completely invariant both in azurin and plastocyanin and may therefore be an obligatory constituent of the copper site. 

In order to understand the charge transfer features of the Blue Copper site, the variable-temperature optical absorption, room-temperature circular dichroism, and magnetic circular dichroism spectra of plastocyanin, stellacyanin, and azurin were studied355. As can be seen for plastocyanin in Fig. 12, the relative intensities (and signs, in the case of CD and MCD) of these transitions vary among the different types of spectra. This is a result of the difference in selection rules for absorption, CD, and MCD spectra, as mentioned in the Introduction. A careful comparison of the three types of spectra and the absorption bandshape temperature dependence (see moment analysis in Ref. 35, pp. 176-177)... 

Details of the position of the —600-nm peak and absorption coefficients for blue copper proteins are indicated in Table I. There are some differences in the peak position, e.g., 597 nm for plastocyanin and 625 nm for azurin (Fig. 6). These may be related to the existence of a fifth ligand in the case of azurin (S), giving rise to trigonal-bipyramidal rather than tetrahedral coordination, but the same trend is not observed with pseudoazurin (12), which also appears to have a weak Cu—O interaction. 

Figure 12 Vibrational enhancement selectivity available from resonance Raman spectroscopy. The UV-visible spectrum of a P. aeruginosa azurin is shown together with two different Raman spectra (frozen solution at 77 K) that derive from laser excitation within theS(Cys) — Cu(II) charge-transfer absorption band at 625nm (647.1 nm) and away from the absorption (488.Onm). Excitation within resonance leads to dramatically increased Raman scattering from the Cu active site, whereas off-resonance excitation produces a spectrum dominated by bands...

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