Investigating the cause of the alkaline transition of phytocyanins

The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His(2)Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pK(a) for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pK(a) for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition.     

Effect of histidine 6 protonation on the active site structure and electron-transfer capabilities of pseudoazurin from Achromobacter cycloclastes

The paramagnetic (1)H NMR spectrum of Cu(II) pseudoazurin [PACu(II)] contains eight directly observed hyperfine-shifted resonances which we have assigned using saturation transfer experiments on a 1:1 mixture of PACu(I) and PACu(II). The spectrum exhibits a number of similarities to those of other cupredoxins, but differences are found concerning the Cu-S(Met) interaction. The spectrum is dependent on pH* in the range 8.5-4.5 (pK(a)* 6.4), and a conformational change involving movement of the copper ion away from the Met toward the equatorial ligands, as a consequence of protonation of the surface His6 residue, is identified. Corresponding changes are also seen in the UV/vis spectrum. The protonation/deprotonation equilibrium of His6 influences the reduction potential of the protein in the same pH range. The self-exchange rate constant of PACu at pH* 6.0 (25 degrees C) is considerably smaller (1.1 x 10(3) M(-1) s(-1)) than the value obtained at pH* 7.6 (3.7 x 10(3) M(-1) s(-1)). The effect on the self-exchange reactivity is mainly due to an alteration in the reorganization energy of the copper site brought about by the structural change resulting from His6 protonation.     

Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant

The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)-->Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A(459)/A(598) ratio of 0.31 for Met16Phe as compared to a A(453)/A(594) ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A C(gamma)H proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic (1)H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an approximately 60 mV increase in the reduction potential of PACu. The pK(a) value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the pi-pi contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH 7.6 (25 degrees C) of 9.8 x 10(3) M(-)(1) s(-)(1) as compared to the considerably smaller value of 3.7 x 10(3) M(-)(1) s(-)(1) for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the pi-pi interaction between His81 and the introduced phenyl ring.     

Spectroscopic characterization of active mutants of manganese peroxidase: mutations on the proximal side affect calcium binding of the distal side.

The mutants at position 242 of manganese peroxidase (MnP), where the native Asp has been substituted with a Ser or a Glu, have been shown to be active, and are here characterized by electronic, EPR, and NMR spectroscopies. We have also mutated another residue on the proximal side, Phe 190 to Val and Leu, yielding active mutants. When studied by the above-mentioned spectroscopies, the mutants at both positions 242 and 190 exhibit three pH-dependent transitions. In contrast to the transitions observed at low and high pH, the spectroscopic studies reveal that the transition at intermediate pH has pK(a) values up to 2 units lower for the mutants at D242E and -S and F190V than for the wild type. This process is due to the ionization of a group that affects the transition to the bis-histidine coordination at the iron. The observed changes in the pK(a) values are related to the altered affinity of the calcium-binding site in the distal pocket. Other variations are observed in the other two pK(a) values. Characterization of the cyanide derivatives indicates that the location and orientation of the distal and proximal His residues are essentially identical to that in the wild type. Our results indicate that mutations on the proximal side residues can affect changes in the distal side. In particular, deprotonation of a group, whose pK(a) is influenced by the nature of the residues in the proximal side, produces a movement of helix B, which in turn induces the coordination of the distal His and the loss of the distal calcium ion.     

pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15)

Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK(a) of 5.7 and a basic limb with pK(a) of 8.8. The acidic limb can be attributed to protonation of a residue affecting k(cat) while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.     

pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone

The effects of pH on the kinetics of association and dissociation of chymotrypsin and the dipeptidyl trifluoromethyl ketone (TFK) N-acetyl-L-leucyl-L-phenylalanyltrifluoromethane (1) were examined through the pH range 4-9.5. The pH dependence of the association rate (kon) is similar to that of kcat/Km for ester and peptide substrates and is dependent on two pK's at 7.0 and 8.9. We assign these pK's to the active site His and to the amino group of the N-terminal isoleucine residue. Ki for the complex of 1 and chymotrypsin has a pH dependence very similar to that of kon, and we conclude that the same ionizable groups which determine the pH dependence of kon are involved. The dissociation constant of the enzyme-inhibitor complex (koff) shows no pH dependence between pH 4 and pH 9.5. The data indicate that the inhibitor reacts with a form of the enzyme in which His 57 is unprotonated, and the resulting complex contains no groups which ionize between pH 4 and pH 9.5. This is consistent with conclusions previously reached from NMR data (Liang & Abeles, 1987). These experiments led to the conclusion that 1 reacts with chymotrypsin to form a tetrahedral complex in which His 57 is protonated (pK greater than 9.5) and the OH group of serine 195 has added to the carbonyl group of 1 to form an ionized hemiketal (pK less than 4.9). The pK of His 57 is increased by greater than 3 units over that in the free enzyme, and the pK of the hemiketal decreased by greater than 4 units compared to the pK in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissecting structural and electrostatic interactions of charged groups in alpha-sarcin. An NMR study of some mutants involving the catalytic residues

The cytotoxic ribonuclease alpha-sarcin is the best characterized member of the ribotoxin family. Ribotoxins share a common structural core, catalytic residues, and active site topology with members of the broader family of nontoxic microbial extracellular RNases. They are, however, much more specific in their biological action. To shed light on the highly specific alpha-sarcin activity, we have evaluated the structural and electrostatic interactions of its charged groups, by combining the structural and pK(a) characterization by NMR of several variants with theoretical calculations based on the Tanford-Kirkwood and Poisson-Boltzmann models. The NMR data reveal that the global conformation of wild-type alpha-sarcin is preserved in the H50Q, E96Q, H137Q, and H50/137Q variants, and that His137 is involved in an H-bond that is crucial in maintaining the active site structure and in reinforcing the stability of the enzyme. The loss of this H-bond in the H137Q and H50/137Q variants modifies the local structure of the active site. The pK(a) values of active site groups H50, E96, and H137 in the four variants have been determined by two-dimensional NMR. The catalytic dyad of E96 and H137 is not sensitive to charge replacements, since their pK(a) values vary less than +/-0.3 pH unit with respect to those of the wild type. On the contrary, the pK(a) of His50 undergoes drastic changes when compared to its value in the intact protein. These amount to an increase of 0.5 pH unit or a decrease of 1.1 pH units depending on whether a positive or negative charge is substituted at the active site. The main determinants of the pK(a) values of most of the charged groups in alpha-sarcin have been established by considering the NMR results in conjunction with those derived from theoretical pK(a) calculations. With regard to the active site residues, the H50 pK(a) is chiefly influenced by electrostatic interactions with E96 and H137, whereas the effect of the low dielectric constant and the interaction with R121 appear to be the main determinants of the altered pK(a) value of E96 and H137. Charge-charge interactions and an increased level of burial perturb the pK(a) values of the active site residues of alpha-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.     

Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy

As a basis for understanding the role of albumin in the transport of metal ions, detailed investigations have been carried out to elucidate the structure of Ni(II)- and Cu(II)-binding site of the peptide residue corresponding to the NH2-terminal peptide fragment 1-24 of human serum albumin by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous medium at different pH values and at different ligand/metal ratios. The results show the following: (i) Diamagnetic Ni(II) complex and paramagnetic Cu(II) complex are in slow exchange NMR time scale. (ii) Titration results of Ni(II)-bound form of peptide 1-24 show the presence of a 1:1 complex in the wide pH range (6.0-11.0), and the same stoichiometry is proposed for Cu(II) as well. (iii) Analysis of the spectra suggests that both Ni(II) and Cu(II) have one specific binding site at the NH2-terminal tripeptide segment (Asp-Ala-His...) involving the Asp alpha-NH2, His N(1) imidazole, two deprotonated peptide nitrogens (Ala NH and His NH), and the Asp COO- group. (iv) Complexation of Ni(II) and Cu(II) causes conformational change near the metal-binding site of the polypeptide chain, but there is no other binding group involved besides those in the first three residues.     

Characterization of Arabidopsis thaliana stellacyanin: a comparison with umecyanin

The cupredoxin domain of a putative type 1 blue copper protein (BCB) from Arabidopsis thaliana was overexpressed and purified. A recursive polymerase chain reaction method was used to synthesize an artificial coding region for the cupredoxin domain of horseradish stellacyanin (commonly known as umecyanin), prior to overexpression and purification. The recombinant proteins were refolded from inclusion bodies and reconstituted with copper, and their in vitro characteristics were studied. Recombinant umecyanin, which is nonglycosylated, has identical spectroscopic and redox properties to the native protein. The UV/Vis and EPR spectra of recombinant BCB and umecyanin demonstrate that they have comparable axial type 1 copper binding sites. Paramagnetic (1)H NMR spectroscopy highlights the similarity between the active site architectures of BCB and umecyanin. The reduction potential of recombinant BCB is 252 mV, compared to 293 mV for recombinant umecyanin. Identical pK(a) values of 9.7 are obtained for the alkaline transitions in both proteins. This study demonstrates that BCB is the A. thaliana stellacyanin and the results form the biochemical basis for a discussion of BCB function in the model vascular plant.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.     

Determining the molecular basis for the pH-dependent interaction between the link module of human TSG-6 and hyaluronan

TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.     

Substrate positioning by His92 is important in catalysis by purple acid phosphatase

Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and/or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala, which eliminates polar interactions of this residue with the active site, resulted in a 10-fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen-bonding interactions, resulted in a three-fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima, with pK(es,1) values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen-bonding network that could fine-tune the position of His92 is consistent with this proposal.     

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin

The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.     

Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N(1)-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N(1)-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k(cat)/K(O2) value changes very little upon mutation with N(1)-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k(cat)/K(M)-pH profiles with N(1)-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK(a) values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK(a) as wild-type Fms1, about ～7.4; this pK(a) is assigned to the substrate N4. The k(cat)/K(O2)-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK(a) of about ～6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k(cat) value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 ?. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure.     

A 1H-NMR study of electronic structure of the active site of Galeorhinus japonicus metmyoglobin

The ferric high-spin form of the myoglobin from the shark Galeorhinus japonicus, which possesses a Gln residue at the distal site instead of the usual His residue, has been studied by 1H-NMR spectroscopy. Using the heme meso-proton (C5H, C10H, C15H and C20H) resonance shift as a diagnostic probe for identifying the coordination system of the iron center in ferric high-spin form of hemoprotein, it has been shown that G. japonicus metmyoglobin (metMb) possesses the pentacoordinated active site. The pH-dependence study of NMR spectra of G. japonicus metMb revealed the appearance of the hydroxyl form of metMb at high pH, indicating that the protein undergoes the transition between the acidic and alkaline forms. The pK value and the rate for this acid-alkaline transition in G. japonicus metMb were found to be approximately 10 and much less than 4 x 10(2) s-1, respectively. Since the pK value of the acid-alkaline transition for the pentacoordinated heme in Aplysia limacina metMb is 7.8 [Giacometti, G.M., Das Ros, A., Antonini, E. & Brunori, M. (1975) Biochemistry 14, 1584-1588] and that of the hexacoordinated heme in sperm whale metMb is 9.1 [Brunori, M., Antonini, E., Fasella, P., Wyman, J. & Rossi-Fanelli, A. (1968) J. Mol. Biol. 34, 497-504], the OH- affinity of the ferric heme iron does not appear to depend on its coordination system. The acid-alkaline transition rate in A. limacina metMb was reported to be much less than 1.5 x 10(2) s-1 [Pande, U., La Mar, G.N., Lecomte, J.T.J., Ascoli, F., Brunori, M., Smith, K.M., Pandey, R.K., Parish, D.W. & Thanabal, V. (1986) Biochemistry 25, 5638-5646] and therefore a slow transition rate may be unique to the pentacoordinated active site of Mb.     

UV Raman monitoring of histidine protonation and H-(2)H exchange in plastocyanin

UV resonance Raman bands of Cu-bound and protonated histidine residues have been detected in (2)H(2)O solutions of poplar plastocyanin. For the Cu(II) protein, slow NH-(2)H exchange of the His37 ligand was monitored via the growth of bands at 1389 and 1344 cm(-1) when Pcy was exchanged into (2)H(2)O, or via their diminution when the protein was exchanged back into H(2)O; the rate constant is 7 x 10(-4)/s at pH (p(2)H) 7.4 at room temperature. The slow exchange is attributed to imidazole H-bonding to a backbone carbonyl. Nearby bands at 1397 and 1354 cm(-1), appear and disappear within the mixing time, and are assigned to the solvent-exposed His87 ligand. The approximately 10 cm(-1) differences between His37 and His87 are attributed to the effect of H-bonding on the imidazole ring modes. The UVRR spectra of the Cu(I) protein in (2)H(2)O reveal a 1408 cm(-1) band, characteristic of NH-(2)H-exchanged histidinium, which grows in as the p(2)H is lowered. Its intensity follows a titration curve with pK(a)=4.6. This protonation is assigned to the His87 residue, whose bond to the Cu(I) is known from crystallography to be broken at low pH. As the 1408 cm(-1) band grows, a band at 1345 cm(-1) diminishes, while another, at 1337 cm(-1) stays constant. These are assigned to modes of bound His87 and His37, respectively, shifted down 7-9 cm(-1) from their Cu(II) positions.     

Reassignment of the active site histidines in ribonuclease A by selective deuteration studies.

The C₂H resonance of the active site histidine residue designated AS-2, which has the lower pK_a of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP-3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS-2 is absent in the spectrum of RNase S′ reconstituted using S-peptide deuterated at the C₂ of His 12, and in that of the RNase S′-CMP-3′ complex. We thus demonstrate that histidine AS-2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C₂H resonances made earlier by other authors.     

Role of copper in folding and stability of cupredoxin-like copper-carrier protein CopC

CopC is a periplasmic copper carrier that, in contrast to cytoplasmic copper chaperones, has a beta-barrel fold and two metal-binding sites distinct for Cu(II) and Cu(I). The copper sites are located in each end of the molecule: the Cu(I) site involves His and Met coordination whereas the Cu(II) site consists of charged residues. To reveal biophysical properties of this protein, we have explored the effects of the cofactors on CopC unfolding in vitro. We demonstrate that Cu(II) coordination affects both protein stability and unfolding pathway, whereas Cu(I) has only a small effect on stability. Apo-CopC unfolds in a two-state reaction between pH 4 and 7.5 with maximal stability at pH 6. In contrast, Cu(II)-CopC unfolds in a three-state reaction at pH6 that involves a partly folded intermediate that retains Cu(II). This intermediate exhibits high thermal and chemical stability. Unique energetic and structural properties of different metalated CopC forms may help facilitate metal transport to many partners in vivo.     

Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His(2)Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N(epsilon2)H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.     

The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin

In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.