Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes

A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants.     

Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.

The heme active site structure of an engineered cytochrome c peroxidase [MnCcP; see Yeung, B. K., et al. (1997) Chem. Biol. 4, 215-221] that closely mimics manganese peroxidase (MnP) has been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CepsilonH shift of the proximal histidine and (15)N shift of the bound C(15)N(-) suggest a weaker heme Fe(III)-N(His) bond in MnCcP compared to WtCcP. Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)-N(His) bond strength of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Temperature,pH, and solvent isotope dependent properties of the active sites of resting-state and cyanide-ligated recombinant cytochrome c peroxidase (H52L) revealed by proton hyperfine resonance spectra

Comparative proton NMR studies have been carried out on high-spin and low-spin forms of recombinant native cytochrome c peroxidase (rCcP) and its His52 --> Leu variant. Proton NMR spectra of rCcP(H52L) (high spin) and rCcP(H52L)CN (low spin) reveal the presence of multiple enzyme forms in solution, whereas only single enzyme forms are found in spectra of wild-type and recombinant wild-type CcP and CcPCN near neutral pH. The spectroscopic behaviors of these forms have been studied in detail when pH, temperature, and solvent isotope composition were varied. For resting-state rCcP(H52L) the comparatively large NMR line widths compromise resolution, but two specific enzyme forms were found. They were interconvertible on the basis of varying temperature. For rCcP(H52L)CN four magnetically distinct enzyme forms were identified by NMR. It was found that these forms dynamically interconvert with changing pH, temperature, and solvent isotope composition (percent D(2)O). These studies have identified the alkaline titration of His52 and essentially identical alkaline enzyme forms for natWTCcPCN and rCcP(H52L)CN. From this work we interpret an essential role of His52 in CcP function to be preservation of a single active site structure in addition to the critical role of general base catalysis.     

Yeast cytochrome c peroxidase: mechanistic studies via protein engineering

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme that catalyzes the reduction of hydrogen peroxide to water by ferrocytochrome c. It was the first heme enzyme to have its crystallographic structure determined and, as a consequence, has played a pivotal role in developing ideas about structural control of heme protein reactivity. Genetic engineering of the active site of CcP, along with structural, spectroscopic, and kinetic characterization of the mutant proteins has provided considerable insight into the mechanism of hydrogen peroxide activation, oxygen-oxygen bond cleavage, and formation of the higher-oxidation state intermediates in heme enzymes. The catalytic mechanism involves complex formation between cytochrome c and CcP. The cytochrome c/CcP system has been very useful in elucidating the complexities of long-range electron transfer in biological systems, including protein-protein recognition, complex formation, and intracomplex electron transfer processes.     

pH Dependence of heme iron coordination, hydrogen peroxide reactivity, and cyanide binding in cytochrome c peroxidase(H52K)

Replacement of the distal histidine, His-52, in cytochrome c peroxidase (CcP) with a lysine residue produces a mutant cytochrome c peroxidase, CcP(H52K), with spectral and kinetic properties significantly altered compared to those of the wild-type enzyme. Three spectroscopically distinct forms of the enzyme are observed between pH 4.0 and 8.0 with two additional forms, thought to be partially denatured forms, making contributions to the observed spectra at the pH extremes. CcP(H52K) exists in at least three, slowly interconverting conformational states over most of the pH range that was investigated. The side chain epsilon-amino group of Lys-52 has an apparent pK(a) of 6.4 +/- 0.2, and the protonation state of Lys-52 affects the spectral properties of the enzyme and the reactions with both hydrogen peroxide and HCN. In its unprotonated form, Lys-52 acts as a base catalyst facilitating the reactions of both hydrogen peroxide and HCN with CcP(H52K). The major form of CcP(H52K) reacts with hydrogen peroxide with a rate approximately 50 times slower than that of wild-type CcP but reacts with HCN approximately 3 times faster than does the wild-type enzyme. The major form of the mutant enzyme has a higher affinity for HCN than does native CcP.     

Oxidation of the His-52 --> Leu mutant of cytochrome c peroxidase by p-nitroperoxybenzoic acid: role of the distal histidine in hydroperoxide activation

Both cytochrome c peroxidase (CcP) and a mutant cytochrome c peroxidase in which the distal histidine has been replaced by leucine, CcP(H52L), are converted to hydroxy-ligated derivatives at alkaline pH. In CcP, the hydroxy-ligated derivative is subsequently converted to a bis-imidazole species prior to protein denaturation while the initial hydroxy-ligated CcP(H52L) is converted to a second, spectroscopically distinct hydroxy-ligated species prior to denaturation. The spectra of the alkaline forms of CcP and CcP(H52L) have been determined between 310 and 700 nm. The pH dependence of the rate of reaction between CcP(H52L) and hydrogen peroxide has been extended to pH 10. The hydroxy-ligated form of CcP(H52L) reacts with hydrogen peroxide 4 times more rapidly than the pentacoordinate, high-spin form of CcP(H52L) that exists at neutral pH. The rate of the reaction between p-nitroperoxybenzoic acid and CcP(H52L) has been measured between pH 4 and pH 8. Neutral p-nitroperoxybenzoic acid reacts with CcP(H52L) 10(5) times more slowly than with CcP while the negatively charged p-nitroperoxybenzoate reacts with CcP(H52L) 10(3) times more slowly than with CcP. These data indicate that the role of the distal histidine during the initial formation of the peroxy anion/heme iron complex is not simply base catalysis.     

Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235----Asn-235 mutant

Proton NMR spectra of cytochrome c peroxidase (CcP) isolated from yeast (wild type) and two Escherichia coli expressed proteins, the parent expressed protein [CcP(MI)] and the site-directed mutant CcP(MI,D235N) (Asp-235----Asn-235), have been examined. At neutral pH and in the presence of only potassium phosphate buffer and potassium nitrate, wild-type Ccp and CcP(MI) demonstrate nearly identical spectra corresponding to normal (i.e., "unaged") high-spin ferric peroxidase. In contrast, the mutant protein displays a spectrum characteristic of a low-spin form, probably a result of hydroxide ligation. Asp-235 is hydrogen-bonded to the proximal heme ligand, His-175. Changing Asp-235 to Asn results in alteration of the pK for formation of the basic form of CcP. Thus, changes in proximal side structure mediate the chemistry of the distal ligand binding site. All three proteins bind F-, N3-, and CN- ions, although the affinity of the mutant protein (D235N) for fluoride ion appears to be much higher than that of the other two proteins. Analysis of proton NMR spectra of the cyanide ligated forms leads to the conclusion that the mutant protein (D235N) possesses a more neutral proximal histidine imidazole ring than does either wild-type CcP or CcP(MI). It confirms that an important feature of the cytochrome c peroxidase structure is at least partial, and probably full, imidazolate character for the proximal histidine (His-175).     

Temperature, pH, and solvent isotope effects on cytochrome c peroxidase mutant N82A studied by proton NMR

The mutant of baker's yeast cytochrome c peroxidase-CN with Ala82 in place of Asn82, [N82A]CcPCN, exhibits a complex solution behavior featuring dynamic interconversion among three enzyme forms that so far have only been detected by NMR spectroscopy. Proton NMR studies of [N82A]CcPCN reveal resonances from each of the three enzyme forms and show that the interconversion among forms is controlled by the pH, temperature, and isotope composition (H₂O vs. D₂O) of the buffer solution. No evidence for a key hydrogen bond between His52 and heme-coordinated cyanide is found in any of the enzyme forms, indicating that disruption of the extensive distal hydrogen bonding network is the source of this phenomenon.     

Paramagnetic properties of the low- and high-spin states of yeast cytochrome c peroxidase

Here we describe paramagnetic NMR analysis of the low- and high-spin forms of yeast cytochrome c peroxidase (CcP), a 34 kDa heme enzyme involved in hydroperoxide reduction in mitochondria. Starting from the assigned NMR spectra of a low-spin CN-bound CcP and using a strategy based on paramagnetic pseudocontact shifts, we have obtained backbone resonance assignments for the diamagnetic, iron-free protein and the high-spin, resting-state enzyme. The derived chemical shifts were further used to determine low- and high-spin magnetic susceptibility tensors and the zero-field splitting constant (D) for the high-spin CcP. The D value indicates that the latter contains a hexacoordinate heme species with a weak field ligand, such as water, in the axial position. Being one of the very few high-spin heme proteins analyzed in this fashion, the resting state CcP expands our knowledge of the heme coordination chemistry in biological systems.     

NMR study of the active site of resting state and cyanide-inhibited lignin peroxidase from Phanerochaete chrysosporium. Comparison with horseradish peroxidase

One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting state and the low spin, cyanide-inhibited derivative of isozyme H2 of the lignin peroxidase, LiP, from Phanerochaete chrysosporium strain BKM 1767. One-dimensional NMR revealed a resting state LiP that is five coordinate at 25 degrees C with an electronic structure similar to that of horseradish peroxidase, HRP. Differential paramagnetic relaxivity was used to identify the C beta H signals of the axial His177. A combination of bond correlation spectroscopy and nuclear Overhauser effect spectroscopy of cyanide-inhibited LiP (LiP-CN) has allowed the assignment of all resolved heme resonances without recourse to isotope labeling, as well as those of the proximal His177 and the distal His48. The surprising effectiveness of the two dimensional NMR methods on such a large and paramagnetic protein indicates that such two dimensional experiments can be expected to have major impact on solution structure determination of diverse classes of heme peroxidases. The two dimensional NMR data of LiP-CN reveal a heme contact shift pattern that reflects a close similarity to that of HRP-CN, including the unusual in-plane trans and cis orientation of the 2- and 4-vinyls. The axial His177 also exhibits the same orientation relative to the heme as in HRP-CN. The proximal His177 contact shifted resonances of both the low spin LiP-CN and high spin LiP are shown to reflect significantly reduced hydrogen bond donation by, or imidazolate character for, the axial histidine in LiP relative to HRP, which may explain the higher redox potential of LiP. The signals are identified for a distal residue that originates from the protonated His48 with disposition relative to the heme similar to that found for the distal His42 in HRP-CN. In contrast, the absence of any resolved signals attributable to an Arg44 in LiP-CN suggest that this distal residue has an altered orientation relative to the heme compared with that of the conserved Arg38 in HRP-CN (Thanabal, V., de Ropp, J. S., and La Mar, G. N. (1987) J. Am. Chem. Soc. 109, 7516-7525).     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: evidence for a single, catalytically active, cytochrome c binding domain

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.     

Spectroscopic characterization and ligand-binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR-1.

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR-1 chlorite dismutase shows two different high-spin ferric heme species, which we have designated 'narrow' (gx,y,z = 6.24, 5.42, 2.00) and 'broad' (gz,y,x = 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co-ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co-ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high-spin and low-spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low-spin EPR signal typical of a five-co-ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, Em(Fe3+/2+) = -23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas-sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.     

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.     

Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants

In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at N epsilon position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at N zeta position). UV-visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. (1)H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the pK(a) of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.     

15N and 1H NMR studies of Rhodospirillum rubrum cytochrome c2

15N-Enriched cytochrome c2 was purified from Rhodospirillum rubrum that had been grown on 15NH4Cl, and the diamagnetic iron(II) form of the cytochrome was studied by 15N and 1H NMR spectroscopy. 15N resonances of the four pyrrole nitrogens, the ligand histidine nitrogens, the highly conserved tryptophan indole nitrogen, and some proline nitrogens are assigned. The resonances of the single nonligand histidine are observed only at low pH because of severe broadening produced by proton tautomerization. The resonances of exchangeable protons bonded to the nitrogens of the ligand histidine, the tryptophan, and some amide groups are also assigned. The exchange rates of the nitrogen-bound protons vary greatly: most have half-lives of less than minutes, the indolic NH of Trp-62 exchanges with a half-time of weeks, and the ligand histidine NH proton exchanges with a half-time of months. The latter observation is indicative of extreme exclusion of solvent from the area surrounding the ligand histidine and lends credence to theories implicating the degree of hydrophobicity in this region as an important factor in adjusting the midpoint potential. The dependence of the 15N and 1H NMR spectra of ferrocytochrome c2 on pH indicates neither the Trp-62 nor the ligand His side chains become deprotonated to any appreciable extent below pH 9.5. The His-18 NH remains hydrogen bonded, presumably to the Pro-19 carboxyl group, throughout the pH titrations. Because neither deprotonated nor non-hydrogen-bonded forms of His-18 are observed in spectra of the ferrocytochrome, the participation of such forms in producing a heterogeneous population having different g tensor values seems unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c-CcP complex [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1-ferrocytochrome c(C102T) as a substrate at pH 7.5. Some of the charge-reversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta- and hexacoordinate heme species in the enzyme. An increase in the amount of hexacoordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the 15 mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c-CcP complex observed in the crystal is the dominant catalytically active complex in solution.     

Effective concentrations of amino acid side chains in an unfolded protein.

Preferential interactions between chain segments are studied in unfolded cytochrome c. The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme. The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence. The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole. When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole. On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region. Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues. These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4). At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein. So one histidine, probably His-18, remains as a heme ligand. The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.     

Proton and nitrogen-15 NMR studies of ferricytochrome c cyanide complexes: remarkable conservation of the heme environment among organisms of diverse origin

The native ferric and cyanide-bound ferric forms of nine vertebrate and two yeast cytochromes c have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy. Spectral comparisons have been made among the cytochromes with emphasis on the signal positions for heme and amino acid ligand protons. Consistent with earlier more limited studies of native ferric cytochromes c, the paramagnetically shifted proton NMR signals show little variation among species with up to 50% substitution of amino acids. Proton NMR spectra for the cyanide complexes also show little variation among species. The nitrogen-15 signal for the coordinated cyanide ion is known to be highly variable among other hemoproteins, but the signal covers a range of only 855 to 865 ppm (nitrate ion reference) for vertebrate cytochromes c and 884 to 886 ppm for yeast cytochromes c. The cyanide ligand probe thus reports an amazing conservation of the heme and proximal ligand environment among the cytochromes. Comparative proton and nitrogen-15 chemical shift values are consistent with a slightly stronger proximal histidine imidazole hydrogen bond to an amino acid carbonyl function than is the case for hemoglobin and myoglobin.