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Interaction-induced Redox Switch in the Electron Transfer Complex Rusticyanin-Cytochrome c 4

1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês

10.1074/jbc.274.43.30365

1083-351X

Marie‐Thérèse Giudici‐Orticoni, Françoise Guerlesquin, Mireille Bruschi, Wolfgang Nitschke,

Electrochemical sensors and biosensors

The blue copper protein rusticyanin isolated from the acidophilic proteobacterium Thiobacillus ferrooxidansdisplays a pH-dependent redox midpoint potential with a pK value of 7 on the oxidized form of the protein. The nature of the alterations of optical and EPR spectra observed above the pK value indicated that the redox-linked deprotonation occurs on the ε-nitrogen of the histidine ligands to the copper ion. Complex formation between rusticyanin and its probable electron transfer partner, cytochrome c 4, induced a decrease of rusticyanin's redox midpoint potential by more than 100 mV together with spectral changes similar to those observed above the pK value of the free form. Complex formation thus substantially modifies the pK value of the surface-exposed histidine ligand to the copper ion and thereby tunes the redox midpoint potential of the copper site. Comparisons with reports on other blue copper proteins suggest that the surface-exposed histidine ligand is employed as a redox tuning device by many members of this group of soluble electron carriers. The blue copper protein rusticyanin isolated from the acidophilic proteobacterium Thiobacillus ferrooxidansdisplays a pH-dependent redox midpoint potential with a pK value of 7 on the oxidized form of the protein. The nature of the alterations of optical and EPR spectra observed above the pK value indicated that the redox-linked deprotonation occurs on the ε-nitrogen of the histidine ligands to the copper ion. Complex formation between rusticyanin and its probable electron transfer partner, cytochrome c 4, induced a decrease of rusticyanin's redox midpoint potential by more than 100 mV together with spectral changes similar to those observed above the pK value of the free form. Complex formation thus substantially modifies the pK value of the surface-exposed histidine ligand to the copper ion and thereby tunes the redox midpoint potential of the copper site. Comparisons with reports on other blue copper proteins suggest that the surface-exposed histidine ligand is employed as a redox tuning device by many members of this group of soluble electron carriers. rusticyanin 4-morpholinepropanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine The vast majority of intramolecular biological electron transfer reactions are now well understood on the basis of Marcus' theory of outer shell electron transfer (1Marcus R.A. Sutin N. Biochim. Biophys. Acta. 1985; 811: 265-322Crossref Scopus (7618) Google Scholar, 2Moser C.C. Keske J.M. Warncke K. Farid R.S. Dutton P.L. Nature. 1992; 355: 796-802Crossref PubMed Scopus (1605) Google Scholar, 3Onuchic J.N. Beratan D.N. Winkler J.R. Gray H.B. Annu. Rev. Biophys. Biomol. Struct. 1992; 21: 340-377Crossref Scopus (371) Google Scholar). In the case ofintermolecular electron transfer the situation is complicated by possible alterations of physico-chemical parameters of the individual redox proteins upon transient formation of an electron transfer complex. Modulations of redox midpoint potentials of either one or both of the involved redox centers upon complex formation have been reported (4Vanderkooi J. Erecinska M. Arch. Biochem. Biophys. 1974; 162: 385-391Crossref PubMed Scopus (34) Google Scholar, 5Dutton P.L. Wilson D.F. Lee C.P. Biochemistry. 1979; 9: 5077-5082Crossref Scopus (258) Google Scholar, 6Moser C.C. Dutton P.L. Biochemistry. 1988; 27: 2450-2461Crossref PubMed Scopus (98) Google Scholar, 7Gray K.A. Davidson V.L. Knaff D.B. J. Biol. Chem. 1988; 263: 13987-13990Abstract Full Text PDF PubMed Google Scholar, 8Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (111) Google Scholar, 9Drepper F. Dorlet P. Mathis P. Biochemistry. 1997; 36: 1418-1427Crossref PubMed Scopus (24) Google Scholar), potentially affecting the driving force of the respective oxidoreduction reactions. Such effects are particularly conspicuous in complexes involving blue copper proteins (8Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (111) Google Scholar, 9Drepper F. Dorlet P. Mathis P. Biochemistry. 1997; 36: 1418-1427Crossref PubMed Scopus (24) Google Scholar, 10Zhu Z. Cunane L.M. Chen Z-W. Durley R.C.E. Mathews F.S. Davidson V.L. Biochemistry. 1998; 37: 17128-17136Crossref PubMed Scopus (100) Google Scholar). For the case of the complex between plastocyanin and photosystem 1, an involvement of the surface-exposed histidine ligand to the copper ion in the modulation of plastocyanin's redox properties was discussed (8Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (111) Google Scholar). The crucial role of the corresponding histidine residue in the amicyanin-methylamine dehydrogenase complex was recently substantiated by site-specific mutagenesis and structural studies (10Zhu Z. Cunane L.M. Chen Z-W. Durley R.C.E. Mathews F.S. Davidson V.L. Biochemistry. 1998; 37: 17128-17136Crossref PubMed Scopus (100) Google Scholar). A unique example of blue copper proteins is provided by rusticyanin (RCy)1, a redox carrier isolated from the extremely acidophilic (pH 2), ore-leaching proteobacterium Thiobacillus ferrooxidans. Rusticyanin owes its name to its astonishingly oxidizing electrochemical potential of +680 mV at pH 2, sufficiently high to oxidize reduced iron compounds. In this work we have identified a tight electron transfer complex between RCy and cytochrome c 4, a further soluble redox protein from T. ferrooxidans. A detailed characterization of this complex with respect to spectral (optical and EPR) and redox properties demonstrated substantial alterations of all these properties in RCy upon complex formation. The correlation between these alterations and respective changes induced in free RCy at high pH values provides a rationale for the effects observed in the complex. A comparison with published data on other blue copper proteins strongly suggests that the mechanism of complex-induced tuning of reduction potentials mediated by the surface exposed histidine is wide spread in this class of proteins. T. ferrooxidans was grown as reported previously (11Nunzi F. Guerlesquin F. Shepard W. Guigliarelli B. Bruschi M. Biochem. Biophys. Res. Commun. 1994; 203: 1655-1662Crossref PubMed Scopus (18) Google Scholar). RCy and cytochrome c 4were purified as described in Nunzi et al. (11Nunzi F. Guerlesquin F. Shepard W. Guigliarelli B. Bruschi M. Biochem. Biophys. Res. Commun. 1994; 203: 1655-1662Crossref PubMed Scopus (18) Google Scholar) and in Cavazza et al. (12Cavazza C. Giudici-Orticoni M.-T. Nitschke W. Appia C. Bonnefoy V. Bruschi M. Eur. J. Biochem. 1996; 242: 308-314Crossref PubMed Scopus (43) Google Scholar), respectively. The dissociation constant of the complex between both proteins was determined using the BiaCore technique (13Giudici-Orticoni M.-T. Nitschke W. Cavazza C. Bruschi M. Biomine. 1997; 97: PB4.1-PB4.10Google Scholar). Optical redox titrations were performed on a Kontron Uvikon 932 spectrophotometer according to Dutton (14Dutton P.L. Biochim. Biophys. Acta. 1971; 226: 63-80Crossref PubMed Scopus (347) Google Scholar) in the presence of ferrocene dicarboxylic acid, ferrocene monocarboxylic acid, and potassium ferricyanide (all at 5 μm). The ambient potential was adjusted with sodium dithionite or potassium hexachloroiridate. EPR spectra were recorded on a Bruker ESP300e X-band spectrometer fitted with an Oxford Instruments liquid helium cryostat and temperature control system. Rusticyanin forms a tight electron transfer complex with cytochrome c 4 . Despite several decades of research, the nature and sequence of electron carriers involved in the initial steps of Fe2+ oxidation by T. ferrooxidans are still uncertain. A number of soluble periplasmic redox proteins potentially participating in this electron transport chain have been purified and characterized and three of them have been studied in more detail, i.e. the blue copper protein rusticyanin (RCy; Ref. 15Cox J.C. Boxer D.H. Biochem. J. 1978; 17: 497-502Crossref Scopus (134) Google Scholar), a high potential iron sulfur protein (Refs.16Kusano T. Takeshima T. Sugawara K. Inoue C. Shiratori T. Yano T. Fukumori Y. Yamanaka T. J. Biol. Chem. 1992; 267: 11242-11247Abstract Full Text PDF PubMed Google Scholar and 17Cavazza C. Guigliarelli B. Bertrand P. Bruschi M. FEMS Microbiol. Lett. 1995; 130: 193-200Crossref Google Scholar) and a diheme cytochrome of thec 4-type (12Cavazza C. Giudici-Orticoni M.-T. Nitschke W. Appia C. Bonnefoy V. Bruschi M. Eur. J. Biochem. 1996; 242: 308-314Crossref PubMed Scopus (43) Google Scholar). Their organization into an electron transport chain bridging the span from Fe2+ to the membrane-bound, energy-conserving electron transport pathways is still a matter of debate (13Giudici-Orticoni M.-T. Nitschke W. Cavazza C. Bruschi M. Biomine. 1997; 97: PB4.1-PB4.10Google Scholar, 18Blake R.C. Shute E.A. Biochemistry. 1994; 33: 9220-9228Crossref PubMed Scopus (62) Google Scholar, 19Yamanaka T. Fukumori Y. Yano T. Kai M. Sato A. Dev. Geochem. 1991; 6: 267-273Google Scholar, 20Ingledew W.J. Cox J.C. Halling P.J. FEMS Microbiol. Lett. 1977; 2: 193-197Crossref Scopus (92) Google Scholar, 21Elbehti A. Nitschke W. Tron P. Michel C. Lemesle-Meunier D. J. Biol. Chem. 1999; 274: 16760-16765Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In order to obtain information concerning the possible electron transfer partners of RCy, we have studied pairwise affinities within the set of soluble redox proteins using the Biacore technique. A particularly strong association constant (K D = 2.07 × 10−7m) was found for the complex of rusticyanin with cytochrome c 4, indicating that electron transfer between these two redox components may represent a genuine segment of the Fe2+-oxidizing pathway. While examining electron transfer reactions between these two redox proteins, we noticed (a) that partially reduced RCy was almost completely oxidized upon addition of ferricytochromec 4 and (b) that cytochromec 4 was reduced by excess of Fe2+ in the presence of RCy, whereas no reduction of cytochromec 4 could be observed when RCy was absent (13Giudici-Orticoni M.-T. Nitschke W. Cavazza C. Bruschi M. Biomine. 1997; 97: PB4.1-PB4.10Google Scholar). Considering the redox midpoint potentials of the free forms of RCy (+590 mV at pH 4.8) and of cytochrome c 4 (+385 mV, +480 mV), these findings strongly indicate significant changes in redox potentials of one or both of these electron transport proteins upon complex formation. Fig.1 shows the redox potential dependence at pH 4.8 of rusticyanin's optical band in the vicinity of 600 nm in the presence of a stoichiometric amount of cytochromec 4. An E m (redox midpoint potential) value of +490 mV was observed, i.e. a value that is by about 100 mV lower than that obtained in free rusticyanin at the same pH value (indicated by the dotted line in Fig. 1). The E m values of both cytochrome c 4 hemes remained constant within experimental precision (not shown). In addition to the drop in electrochemical potential, alterations of optical and EPR parameters of the copper site in rusticyanin were observed in the complex. The peak wavelength of the optical band in the vicinity of 600 nm, typical for blue copper proteins in the oxidized state, was found to be shifted in the complex by 14 nm toward shorter wavelengths (Fig.2, continuous versus dashed trace). This spectral band arises from a charge transfer transition between the copper ion and the sulfur atom of the cysteine ligand (22Solomon E.I. Baldwin M.J. Lowery M.D. Chem. Rev. 1992; 92: 521-542Crossref Scopus (903) Google Scholar), and modifications of this band are therefore indicative of substantial rearrangements of the electron density distribution between the copper site and its ligands. Fig.3, trace b, shows the X-band EPR spectrum recorded on oxidized free RCy at a concentration of 20 μm. The obtained spectrum corresponded to those previously reported for oxidized RCy at low pH values (Ref. 23Cox J.C. Aasa R. Malmström B.G. FEBS Lett. 1978; 93: 157-160Crossref PubMed Scopus (45) Google Scholar, see also Fig. 3, trace c). Addition of a stoichiometric amount of oxidized cytochrome c 4 to the sample used for recording the spectrum shown in Fig. 3, trace b, resulted in dramatically altered EPR spectral properties (Fig. 3, trace a) involving an increase in g∥ as well as modifications in apparent hyperfine parameters on all g values. These EPR spectra thus provided evidence for redistributions of the unpaired electron density at the copper site upon complex formation corroborating the significantly altered electronic structure of the copper-ligand sphere in the complex.Figure 3EPR spectra of oxidized RCy prior to (trace b) and after (trace a) complex formation with cytochrome c 4 at pH 4.8 as well as those of free RCy at pH values of 4.3 (trace c), 8.5 (trace d), and 9.5 (trace e). Complete oxidation of the sample was achieved by addition of iridium chloride followed by removal of the oxidant via passage through a PD-10 column. The sample contained 100 μm rusticyanin in ammonium acetate, pH 4.8; MOPS (pH 7); glycyl-glycyl, pH 8.2; Tricine-NaOH, pH 9; or 20 μmcytochrome c 4 and 20 μmrusticyanin in 20 mm ammonium acetate, pH 4.8. For better comparison, EPR spectra of the complex and of free RCy at different pH values were normalized to comparable signal amplitudes. The inner part of the spectrum (in between vertical dotted lines) was divided by 4. Instrument settings: temperature, 20 K; microwave frequency, 9.43 GHz; microwave power, 6.7 milliwatts; modulation amplitude, 0.5 millitesla.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The changes in the optical spectrum induced by complex formation was suggestively reminiscent of previously reported data on rusticyanin obtained at pH values above 7,i.e. a blueshift of the charge transfer band from 597 to 577 nm (11Nunzi F. Guerlesquin F. Shepard W. Guigliarelli B. Bruschi M. Biochem. Biophys. Res. Commun. 1994; 203: 1655-1662Crossref PubMed Scopus (18) Google Scholar). Apart from the blueshift, the charge transfer band was seen to persist up to pH values of about 10, demonstrating that the overall distorted tetrahedral ligand geometry (involving one cysteine-, one methionine, and two histidine residues (24Botuyan M.V. Toy-Palmer A. Chang J. Blake R.C., II Beroza P. Case D.A. Dyson H.J. J. Mol. Biol. 1996; 263: 752-767Crossref PubMed Scopus (88) Google Scholar, 25Walter R.L. Ealick S.E. Friedman A.M. Blake R.C., II Proctor P. Shoham M. J. Mol. Biol. 1996; 263: 730-751Crossref PubMed Scopus (108) Google Scholar), see Fig. 6) was maintained up to this pH value. Above pH 10, an essentially irreversible loss of the Cu-SCys charge transfer transition was observed indicating denaturing of the protein. The copper center in rusticyanin thus retains its ligand scheme up to high pH values, whereas the copper-ligand interactions undergo noticeable alterations in the pH range between 7 and 10 reflected by the blueshift of the charge transfer band. The similarity of the spectral changes observed at high pH and in the complex suggested a possible correlation of both phenomena. To substantiate such a correlation, we examined the pH dependence of rusticyanin's EPR spectral parameters and of its reduction potential in the alkaline region. EPR spectra recorded at several pH values between pH 4 and pH 11 (Fig. 3, traces c–e) indeed showed that new spectral forms with significantly altered g and hyperfine tensors appear above pH 7. It is of note that comparable spectral transitions at high pH values have already been reported previously (23Cox J.C. Aasa R. Malmström B.G. FEBS Lett. 1978; 93: 157-160Crossref PubMed Scopus (45) Google Scholar); the much lower signal-to-noise ratio, however, precluded an interpretation of the EPR parameters in the alkalinic region. The spectra obtained in the region between pH 7 and pH 10 (Fig. 3,traces c–e) were heterogeneous, i.e. reflected the sequential deprotonations of more than one group. The general spectral features of the high pH paramagnetic species, however, involved a shift of g∥ toward higher values and the loss of the hyperfine structure on g⊥, i.e.intriguingly resembling those observed in the complex. At pH 9.5, the new paramagnetic species have become dominant, whereas the low pH spectrum has essentially vanished (as judged, for instance, from the disappearance of the trough at g = 1.96 in Fig. 3). Fig.4 shows a pH titration of signals characteristic for the low and the high pH spectra, respectively. As can be seen from this figure, the low pH spectrum converted into the high pH spectra in the pH range between 7 and 9. Above pH 9.5, a progressive loss of the signal was found most probably corresponding to the loss of the copper site as discussed above. The redox midpoint potential of rusticyanin has previously been reported to show only a weak pH dependence up to pH 6.2 (26Haladjian J. Nunzi F. Bianco P. Bruschi M. J. Electroanal. Chem. 1993; 352: 329-335Crossref Scopus (16) Google Scholar). Stimulated by the above described phenomena observed at high pH values, we extended the determination of rusticyanin's E m values up to pH 9. Fig.5 shows the results of equilibrium redox titrations of free RCy between pH 3 and pH 9. The weak pH dependence seen at low pH values ceased at about pH 7 and was replaced by a strongly pH-dependent curve with a slope of about 60 mV/pH above this pK value. The obtained dependence at high pH values is characteristic of the involvement of a dissociable proton on the oxidized form of the protein in the redox transition. At pH 9, theE m value of free RCy corresponds roughly to that observed at pH 4.8 in the complex with cytochromec 4 (see Fig. 5). Since the slope of theE m versus pH curve was still close to the theoretical value for a single redox-linked proton (−59 mV/pH) at pH 9, the pK of the dissociable proton on the reduced form of the protein must be significantly above this pH value. A strikingly similar pH dependence of redox properties is observed in the [2Fe-2S] protein from cytochrome bc complexes, the so-called Rieske proteins. The [2Fe-2S] cluster in this class of proteins is ligated by two cysteine and two histidine residues. The Rieske clusters are characterized by a pH-dependent redox midpoint potential above a pK value that varies in the range of pH 6 to pH 8 between species (27Schoepp B. Brugna M. Lebrun E. Nitschke W. Adv. Inorg. Chem. 1999; 47: 335-360Crossref Scopus (13) Google Scholar, 28Brugna M. Nitschke W. Asso M. Guigliarelli B. Lemesle-Meunier D. Schmidt C. J. Biol. Chem. 1999; 274: 16766-16772Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The redox-linked proton is generally believed to correspond to the nitrogen proton of the imidazole moiety of the histidine ligands (27Schoepp B. Brugna M. Lebrun E. Nitschke W. Adv. Inorg. Chem. 1999; 47: 335-360Crossref Scopus (13) Google Scholar). A similar situation appears to apply to RCy. The only residues that are liable to have pK values in the observed range, and the deprotonation of which will induce significant modifications of the electronic structure at the copper ion, are the two histidine ligands His-85 and His-143 (see Fig.6). The additional negative charges on the histidinates above the respective pK values would be expected to render the copper ion more difficult to reduce and thus to lower its redox midpoint potential. The sequential deprotonation of the two histidine ligands at high pH values also rationalizes the observed heterogeneity of EPR spectra recorded in the alkaline region. The spectra shown in Fig. 3, traces d and e, represent superpositions of spectra arising from RCy molecules with either one or both histidine ligands in the deprotonated state. The ensemble of the similarities between RCy at high pH and in the complex with cytochrome c 4demonstrates that docking of RCy to cytochromec 4 induced changes at the copper site related to the redox-linked deprotonation at high pH values. Since the respective effects in the complex were observed at pH 4.8 and below, complex formation must have decreased the pK value of the redox-linked proton by about 4 pH units (see above). From an inspection of the three-dimensional structure of RCy (24Botuyan M.V. Toy-Palmer A. Chang J. Blake R.C., II Beroza P. Case D.A. Dyson H.J. J. Mol. Biol. 1996; 263: 752-767Crossref PubMed Scopus (88) Google Scholar) (see Fig. 6), the Nε proton on the exposed copper-ligand histidine 143 appears predestined to form hydrogen bridges with suitable residues on its partner in complex formation. Such an interaction would weaken the H–Nε (His-143) bond (i.e. decrease the pK value of the protonation site) and thereby induce electronic changes in the imidazole ring resembling deprotonation (Fig. 6). Attempts to co-crystallize RCy and cytochrome c 4 in order to obtain a detailed picture of the interaction surface in the complex are presently under way in our laboratory. The observed complex-induced alterations of RCy's redox properties make sense with regard to the thermodynamic constraints of electron transfer in Thiobacilli. These organisms use the relatively weak reducing power of the iron(II) (E m,2 ≈ 700 mV, Ref. 29Ingledew W.J. Biochim. Biophys. Acta. 1982; 683: 89-117Crossref PubMed Scopus (294) Google Scholar), abundant in the growth medium, to drive electron transport toward molecular oxygen (E m,2 ≈ 1150 mV, Ref. 29Ingledew W.J. Biochim. Biophys. Acta. 1982; 683: 89-117Crossref PubMed Scopus (294) Google Scholar) via a membrane-bound cytochrome oxidase (Fig.7). From comparisons to other organisms (30Ng T.C.N. Laheri A.N. Maier R.J. Biochim. Biophys. Acta. 1995; 1230: 119-129Crossref PubMed Scopus (19) Google Scholar, 31Pettigrew G.W. Brown K.R. Biochem. J. 1988; 252: 427-435Crossref PubMed Scopus (32) Google Scholar) and from the operon structure of the cytochromec 4/oxidase genes (32Appia-Ayme C. Bengrine A. Cavazza C. Giudici-Orticoni M.-T. Bruschi M. Chippaux M. Bonnefoy V. FEMS Microbiol. Lett. 1998; 167: 171-177PubMed Google Scholar), it appears likely that cytochrome c 4 serves as the immediate donor to cytochrome oxidase in Thiobacilli. Since the involvement of RCy in the early steps of Fe(II) oxidation has been shown, electron transport would be expected to proceed from the copper protein toward the cytochrome. Such an electron transport, however, would be strongly hampered by the unfavorable redox equilibrium if there were not the above shown complex-induced drop in reduction potential of RCy (see Fig. 7). The extent of the observed drop inE m value, on the other side, is thermodynamically equivalent to a more than 100-fold preferential binding of the oxidized form of RCy to cytochromec 4, i.e. seemingly in contradiction to electron transfer from the copper protein to the cytochrome. RCy, however, is by far the most abundant soluble redox protein in Thiobacilli accounting for up to 5% of the total soluble protein. This large pool of RCy molecules will be held at a very high degree of reduction by the abundant Fe(II) in the medium, assuring saturation of the binding sites on cytochrome c 4 by reduced RCy and thus allowing efficient electron transfer from RCy to cytochrome c 4. As shown in Fig. 6, the likely key residue mediating the interaction-induced changes in electrochemical potential is located in a strikingly strategic position on the surface of the protein. This histidine residue in its privileged position is conserved in the large majority of blue copper proteins (33Nunzi F. Woudstra M. Campèse D. Bonicel J. Morin D. Bruschi M. Biochim. Biophys. Acta. 1993; 1162: 28-34Crossref PubMed Scopus (44) Google Scholar). Modifications of redox properties reminiscent of those seen for RCy have furthermore been observed for the electron transfer complexes between amicyanin and methylamine dehydrogenase (7Gray K.A. Davidson V.L. Knaff D.B. J. Biol. Chem. 1988; 263: 13987-13990Abstract Full Text PDF PubMed Google Scholar, 10Zhu Z. Cunane L.M. Chen Z-W. Durley R.C.E. Mathews F.S. Davidson V.L. Biochemistry. 1998; 37: 17128-17136Crossref PubMed Scopus (100) Google Scholar) and between plastocyanin and photosystem I (8Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (111) Google Scholar). The crucial role of the surface-exposed histidine in complex-induced alterations of redox midpoint potential of the copper site has recently been demonstrated for the electron transfer complex between amicyanin and methylamine dehydrogenase (10Zhu Z. Cunane L.M. Chen Z-W. Durley R.C.E. Mathews F.S. Davidson V.L. Biochemistry. 1998; 37: 17128-17136Crossref PubMed Scopus (100) Google Scholar, 34Chen L. Durley R.C. Mathews F.S. Davidson V.L. Science. 1994; 264: 86-90Crossref PubMed Scopus (209) Google Scholar). Free amicyanin shows a pH-dependent redox potential below pH 7.5 and constantE m values above this pK value, indicating a redox-linked protonation on the reduced form of the protein. The presence of such a pK value on the reduced protein is common to the large majority of blue copper proteins (35Lommen A. Canters G.W. J. Biol. Chem. 1990; 265: 2768-2774Abstract Full Text PDF PubMed Google Scholar). Already more than a decade ago, the protonation of the δ-nitrogen on the exposed histidine ligand has been identified as the molecular basis for the respective redox phenomenon in plastocyanin (36Guss J.M. Harrowell P.R. Murata M. Norris V.A. Freeman H.C. J. Mol. Biol. 1986; 192: 361-387Crossref PubMed Scopus (386) Google Scholar). This protonation entails breakage of this histidine-copper ligation and subsequent rotation of the histidine side chain (36Guss J.M. Harrowell P.R. Murata M. Norris V.A. Freeman H.C. J. Mol. Biol. 1986; 192: 361-387Crossref PubMed Scopus (386) Google Scholar). The recent study of the amicyanin-methylamine dehydrogenase complex has shown that a corresponding rotation of the exposed histidine in amicyanin is hampered by the presence of methylamine dehydrogenase at the interaction surface of the complex (10Zhu Z. Cunane L.M. Chen Z-W. Durley R.C.E. Mathews F.S. Davidson V.L. Biochemistry. 1998; 37: 17128-17136Crossref PubMed Scopus (100) Google Scholar). The resulting stabilization of the copper-histidine bond is equivalent to a decreased pK value of the protonation site on Nδ and therefore translates into a decrease of amicyanin's redox midpoint potential at pH values below the pK of the free form (10Zhu Z. Cunane L.M. Chen Z-W. Durley R.C.E. Mathews F.S. Davidson V.L. Biochemistry. 1998; 37: 17128-17136Crossref PubMed Scopus (100) Google Scholar). Rusticyanin, by contrast, is an acid-stable protein and the pK value of the Nδ protonation site therefore needs to be shifted to pH values significantly below pH 2 in order to assure integrity of the copper site at the physiological pH values of the organism (24Botuyan M.V. Toy-Palmer A. Chang J. Blake R.C., II Beroza P. Case D.A. Dyson H.J. J. Mol. Biol. 1996; 263: 752-767Crossref PubMed Scopus (88) Google Scholar, 25Walter R.L. Ealick S.E. Friedman A.M. Blake R.C., II Proctor P. Shoham M. J. Mol. Biol. 1996; 263: 730-751Crossref PubMed Scopus (108) Google Scholar). Consequently, the above described pK value on the reduced form of the protein was not observed in RCy (26Haladjian J. Nunzi F. Bianco P. Bruschi M. J. Electroanal. Chem. 1993; 352: 329-335Crossref Scopus (16) Google Scholar). The drastically decreased pK value on Nδ (His-143) most probably results from the presence of the two aromatic amino acid residues Phe-83 and Phe-51 in the immediate vicinity of the surface-exposed histidine 143 (see Fig.6), rendering the environment of this residue significantly more hydrophobic in RCy than in other blue copper proteins. However, the reduced solvent accessibility of the surface-exposed histidine obviously also affects the pK-value of the proton on Nε. This pK is found slightly above pH 7 in RCy (Fig. 5), whereas it is beyond the experimentally accessible range of pH values in other blue copper proteins. In RCy, it is, therefore, a pK shift of the Nε proton that lends itself for inducing changes in redox midpoint potential upon complex formation rather than a respective shift of NδH, as described for the amicyanin-methylamine dehydrogenase complex (10Zhu Z. Cunane L.M. Chen Z-W. Durley R.C.E. Mathews F.S. Davidson V.L. Biochemistry. 1998; 37: 17128-17136Crossref PubMed Scopus (100) Google Scholar). It therefore appears likely to us that the surface-exposed histidine ligand to the copper ion is employed as a redox tuning device in blue copper proteins in general. We thank G. Canters (Leiden, The Netherlands), F. Drepper (Freiburg, Federal Republic of Germany), M. Hippler (Geneva, Switzerland), A. G. Sykes (Newcastle, UK), and R. J. P. Williams (Oxford, UK) for stimulating discussions and communicating results prior to publication; R. Toci (Marseille, France) for growing the bacterial cells; and P. Bertrand (Marseille, France) for access to the EPR facilities.