Azurin derivatives

Table 6 Ligand vibrations of P. aeruginosa azurin derivatives ...

D.R. McMillin, Purdue University In addition to the charge effects discussed by Professor Sykes, I would like to add that structural effects may help determine electron transfer reactions between biological partners. A case in point is the reaction between cytochrome C551 and azurin where, in order to explain the observed kinetics, reactive and unreactive forms of azurin have been proposed to exist in solution (JL). The two forms differ with respect to the state of protonation of histidine-35 and, it is supposed, with respect to conformation as well. In fact, the lH nmr spectra shown in the Figure provide direct evidence that the nickel(II) derivative of azurin does exist in two different conformations, which interconvert slowly on the nmr time-scale, depending on the state of protonation of the His35 residue (.2) As pointed out by Silvestrini et al., such effects could play a role in coordinating the flow of electrons and protons to the terminal acceptor in vivo. 

In a sense, the comparative studies that were performed in the ruthenium derivatives of plastocyanin and azurin are also related to this kind of approach [152]. 

Fig. 6. Paramagnetic enhancements to water NMRD profiles for solutions of wild type azurin ( ), and its Hisll7Gly (O) and His46Gly ( ) derivatives at 278 K and pH 7.5 (32).

Spectroscopic studies of Co (II) derivatives of stellacyanin, plastocyanin, and azurin have established that the charge transfer interpretation is preferred (10, 11). Intense bands (c 2 X 103) that appear to be analogous to the 600-nm system of blue proteins are observed between 300 and 350 nm in the Co (II) derivatives. The shift in band position of about 16 kK [Cu(II) << Co (II)] accords well with expectation for an LMCT transition. The visible and near-infrared absorption, CD, and MCD spectra of Co (II) derivatives of stellacyanin, plastocyanin, and azurin have been interpreted (12) successfully in terms of the d-d transitions expected for distorted tetrahedral metal centers (Table I). Average ligand field parameters are the same for all three Co (II) proteins (Dq = 490, B = 730 cm"1), which strongly suggests that the donor atom... 

Intriguingly, the blue copper sites, especiaUy those with a carbonyl oxygen at the axial coordination position, display high affinity for Zn + ions. Mutants in which the Met is replaced by Gin or Glu preferentiaUy bind Zn + when expressed in heterologous systems, e.g., Escherichia coli. Examples include azurin, amicyanin, nitrite reductase, and possibly also plastocyanin (Diederix et al., 2000 Hibino et al., 1995 Murphy et al., 1995 Nar et al., 1992a Romero et al., 1993). In the case of azurin it has been shown that both wild-type and the Met—Gin mutant have the same affinity for both Zn +and Cu + (Romero ci a/., 1993). In addition, EXAFS studies showed that some preparations of blue copper proteins purihed from their natural sources also contain small fractions of Zn derivatives (DeBeer George, personal communication). 

Co(II) has been the most useful metal probe for the study of BCE The Co(II) derivatives of Ps. aeruginosa azurin (Moratal Mascarell et al., 1993a Piccioli et al., 1995 Salgado et al., 1995), Rhus vernicifera stellacyanin (Fernandez et al., 1997 Vila, 1994 Vila and Fernandez, 1996), Ac. cyclo-clastes pseudoazurin (Fernandez et al., 2001), Thi. ferrooxidans rusticyanin (Donaire et al., 2001), Thi. versutus amicyanin (Salgado et al., 1999), several mutants of azurin (Piccioli et al., 1995 Salgado et al., 1996, 1998a Vila et al., 1997), and the M99Q mutant of amicyanin (Diederix et al., 2000) have been prepared, and their H NMR spectra have been characterized. 

Figure 27 Room temperature UV-visible spectra of (a) native P. aeruginosa azurin and its (b) Ni(II)-, and (c) Co(ll)-substituted derivatives...

Figure 29 Low-temperature (77 K) RR spectra of P. aeruginosa azurin obtained in the(A) 100-500cm" and (B) 500-1000cm" regions on (a) native Cu(ll) protein (647.1-nm excitation) and (b) its Ni(ll)-substituted derivative (413.1-nm excitation). Asterisks indicate ice Raman bands...

Mizoguchi TJ, Dibilio AJ, Gray HB, Richards JH (1992) Blue to type-2 binding - copper(II) and cobalt(II) derivatives of a Cysl 12Asp Mutant of Pseudomonas aeruginosa azurin. J Am Chem Soc 114 10076-10078... 

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