Pseudomonas aeruginosa cytochrome C551: probing the role of the hydrophobic patch in electron transfer
Introduction
Electron transfer between proteins involves the formation of a complex in which the partners assemble (transiently) through complementary contact surfaces, and the donor–acceptor electron flow is modulated by the intervening medium separating the redox centers [1]. In some cases, such as the photosynthetic reaction centers and the mitochondrial electron transport complexes, the subunit assemble in a stable oligomer whose structure may be obtained by crystallography [2], [3], [4], [5]. On the other hand, in the case of soluble electron carrier proteins, rather than association into a single specific configuration, association between redox partners seems to occur through ‘pseudospecific’ surfaces involving hydrophobic contacts stabilized by complementary electrostatic charges [6]. Therefore, many of the physiologically competent electron transfer (ET) complexes are not accessible to crystallographic investigation due to their limited thermodynamic stability which hampers crystallization; only few examples are reported in the literature [7], [8], [9], and interpretation of ET experiments between globular proteins often remains controversial [10]. In some cases NMR is filling the gap, but useful information is limited by the size of the relevant complex and by the relative stability of the association between partners [11], [12], [13]. Modification of residues within the presumed interface between redox partners proved therefore essential to probe the importance of surface complementarity for recognition and this approach makes use of site-directed mutagenesis strategy.
Cytochrome c551 from Pseudomonas aeruginosa (hereinafter Pa-c551) is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of nitrite reductase (NIR) [14]. The functional properties of Pa-c551 have been extensively investigated [15]. The reactions with non-physiological small inorganic redox reactants [16] and with other macromolecules, like blue copper proteins [17], [18], eukaryotic cytochrome c [19] and the physiological partner NIR [18], [20] have provided a test for protein–protein ET. The self-exchange rate, a critical parameter in the framework of Marcus theory [21], was found to be high (k=107 M−1 s−1) [22].
The three-dimensional structure of Pa-c551, which is a member of bacterial class I cytochromes, shows a single low-spin heme with His–Met ligation and the typical polypeptide fold which however leaves the edges of pyrrole rings II and III of the heme exposed [23], [24]. The lack of a 20-residue omega loop, present in the mammalian class I cytochromes, causes further exposure of the heme edge at the level of propionate 13 (HP-13, recommended IUB–IUPAC nomenclature). The distribution of charged residues on the surface of Pa-c551 is very anisotropic: one side is richer in acidic residues (five glutamates and five aspartates) whereas the other displays a ring of positive side chains, mainly lysines, located at the border of a hydrophobic patch which surrounds the heme crevice. This patch comprises residues Gly11, Val13, Ala14, Met22, Val23, Pro58, Ile59, Pro60, Pro62, Pro63 and Ala65 (Fig. 1). The anisotropic charge distribution leads to a large dipolar moment which is important for ET complex formation.
A charge distribution similar to that described above for Pa-c551 has been reported for other periplasmic electron transfer proteins and their electron acceptors whose three-dimensional structure is available [6]. Moreover, modification by site-directed mutagenesis of residues within the hydrophobic or charged patch has shown for different proteins the importance of surface complementarity for binding and ET [among others see [25], [26]]. As an example, evidence for the relevance of the hydrophobic patch for the electron transfer properties of the cuproprotein azurin from P. aeruginosa came from the studies carried out on mutants of residues Met44 and Met64 changed to positively and negatively charged amino acids [27], [28], [29].
In the present work we have introduced specific mutations in the hydrophobic patch of Pa-c551 (see above) and have analysed the effect of these mutations on the self-exchange rate and the electron transfer activity towards P. aeruginosa cd1 NIR (Pa-NIR), the physiological electron acceptor, and P. aeruginosa azurin.
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Bacterial strains and growth
E. coli HB101 was the host strain for plasmid manipulations. Pseudomonas putida strain PaW340, a plasmid free StR, Trp− mutant of mt2, was grown at 30°C, in LB liquid medium. When appropriate, antibiotics were added at the following concentrations (mg/l): ampicillin (Amp) 100, kanamicin (Km) 30. Cultures were grown in the presence of 5 mM meta-toluate (m-Tol) when required for induction of the Pm promoter.
Mutagenesis and protein purification
All DNA manipulations were performed following standard protocols. The construction of
General characterization
In view of the high GC content of P. aeruginosa genes, site-directed mutagenesis of Pa-c551 was obtained by PCR. The mutant proteins were expressed and purified as described in Experimental with yields comparable to those of the wt protein. The absorption spectrum of every mutant is identical to that of the wt in both redox states; moreover the far UV circular dichroism spectrum (200–250 nm), diagnostic of the α-helical content of the protein, is also unchanged (not shown). The thermodynamic
Discussion
The electrostatic and hydrophobic properties of the surface of electron transfer proteins play a crucial role in modulating the rate of ET. Often a marked asymmetry in surface charge distribution, yielding stable dipolar moments important for ET complex formation, is observed. Recent evidence indicates that recognition between soluble redox partners is likely to involve ‘pseudospecific’ surfaces in which hydrophobic interactions are stabilized by complementary electrostatic charges rather than
Concluding remarks
It seems clear from this and other studies that a site-directed mutagenesis strategy provides very useful information in order to map the surface involved in the interaction between proteins in electron transfer complexes. Moreover, we have shown that two mutations with similar physicochemical properties (aspartate and glutamate) introduced in the same region of the surface of a redox protein may exert different effects, as seen comparing the properties of two mutants of Pa-c551 namely V23D and
Abbreviations
- Pa-c551
Cytochrome c551 from Pseudomonas aeruginosa
- ET
Electron transfer
- NIR
Nitrite reductase
Acknowledgements
A grant from the CNR of Italy (Target Project on Biotechnology) is gratefully acknowledged. F.C. was the recipient of a researcher mobility grant from CNR of Italy to perform the NMR measurements at Leiden University (The Netherlands). Dr. Nicolini and Mr. DaGai (Istituto Superiore di Sanità, Rome, Italy) are acknowledged for help in the fermentation of bacterial strains.
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