Distal His Research Articles

Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4°C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP(MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met–Lys–Thr (MKT) at the N-terminus, CCP(MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP(MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP(MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP(MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (≤5%) in the spectrum of CCP(MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP ( λ CT=637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP(MKT) ( λ CT=637 nm) solved at 2.0 Å resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.

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.

Fluoride binding in hemoproteins: the importance of the distal cavity structure

The electronic absorption and resonance Raman spectra of the fluoride complexes of various peroxidases and selected site-directed mutants have been studied at pH 5.0, and compared to the spectra obtained for the myoglobin-F adduct. It is shown that the electronic absorption maxima depend on the degree of conjugation between the porphyrin macrocycle and the vinyl substituents. Moreover, it is confirmed that the wavelength of the CT1 band is a sensitive probe of axial ligand polarity and of its interaction with the distal protein residues. The results highlight the different mechanism of stabilization of the fluoride ligand exerted by the distal residues in myoglobin and peroxidases. In peroxidases, the Arg is determinant in controlling the ligand binding via a strong hydrogen bond between the positively charged guanidinium group and the anion. Mutation of Arg to Leu decreases the stability of the complex by 900-fold, suggesting that this interaction stabilizes the complex by 4 kcal/mol. The distal His also...

Physiology of the escape rhythm after radiofrequency atrioventricular junctional ablation.

The physiology of the escape rhythm (ER) and its response to pharmacological modulation under varying autonomic conditions were studied 48 patients undergoing radiofrequency ablation of the atrioventricular junction (AVJ) for refractory atrial fibrillation. The QRS morphology and cycle length (CL) of the baseline ER were measured 15 minutes postablation. The CL of the ER was measured in response to doses of isoproterenol, atropine, adenosine, lidocaine, and verapamil. The ER QRS was narrow (QRS < 120 ms) in 20 patients and wide (QRS > 120 ms) in 28 patients. Of the 28 patients with wide QRS ER, 11 patients had a new bundle branch block (8 patients new right bundle branch block [RBBB] and 2 patients new left bundle branch block [LBBB]). The ERCL was similar in both narrow and wide ERs (1,593 +/- 376 ms and 1,516 +/- 296 ms, P = 0.44). In 23 patients receiving isoproterenol infusion, the ER CL decreased with increasing doses from 1 mcg/min to 2 mcg/min (1,378 +/- 200 to 1,240 +/- 229 ms, P < 0.001), but did not decrease further at 3 mcg/min (1,201 +/- 192 ms, P = 0.48 vs 2 mg/min). Seven patients received 0.02 mg/kg of atropine, and ER decreased significantly (1,572 +/- 408 ms to 1,319 +/- 333 ms, P = 0.028). In 30 patients who received intravenous boluses of adenosine (6-18 mg), the ER did not change significantly. In 28 patients who received 150 mg of lidocaine, the ER increased from 1,462 +/- 286 ms to 1,715 +/- 467 ms (P < 0.001), and one patient developed transient asystole. Nineteen patients received 7.5 mg of verapamil, and the ER did not change (1,488 +/- 313 ms to 1,513 +/- 666 ms, P = 0.80). There was no significant difference in response to isoproterenol, adenosine, lidocaine, or verapamil between the patients with wide and narrow QRS ERs. We conclude that patients may have stable ERs immediately following AVJ ablation even when a wide complex ER results. The ER is responsive to sympathetic stimulation and vagal blockade. The ER is prolonged after lidocaine but not after verapamil, suggesting response to sodium but not to calcium channel blockade. These data are consistent with an ER originating in the distal compact AV node or proximal His bundle.

Site-directed mutagenesis in hemoglobin: attempts to control the oxygen affinity with cooperativity preserved.

We synthesized three artificial human hemoglobin mutants in which Lys-66(E10)beta was replaced by Ser, Arg or Thr by site-directed mutagenic protein engineering. These engineered hemoglobins were designated as eHb K66betaS, eHb K66betaR and eHb K66betaT, respectively. By synthesizing these mutants we attempted to control the oxygen affinity of hemoglobin with cooperativity preserved and to clarify the functional role of Lys-66(E10)beta, as well. Such attempts may be useful for creating an oxygen carrier suitable as a blood substitute. The oxygen affinities of eHbs K66betaS, K66betaR and K66betaT were 1.3-, 1.5- and 2.3-fold, respectively, lower than that of reconstituted Hb A. Their allosteric properties such as the Bohr effect and the effect of inositol hexaphosphate were well preserved. Since the oxygen affinity of eHb K66betaT is comparable with that of red cells, it may be a potential candidate for a blood substitute. X-Ray crystallographic data for Hb Chico [Lys-66beta-->Thr], which is identical with eHb K66betaT, together with our computer simulation indicate that an interaction between the introduced Thr and the distal His via a water molecule lowers the oxygen affinity for the T state eHb K66betaT.

Electrospray ionization mass spectrometric study of the multiple intracellular monomeric and polymeric hemoglobins of Glycera dibranchiata.

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500-15,200 Da) and an approximately equal number of polymer Hb peaks (15,500-16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; -2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2-4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.

Structureactivity relation of horseradish peroxidases as studied with mutations at heme distal and proximal sites

Abstract

Supernormal conduction in a case of mobitz type II atrioventricular block

A 52-year-old woman exhibited Mobitz type II atrioventricular block with right bundle branch block and 1:1 atrioventricular conduction at or slower than 80 beats/min. Electrophysiologic study revealed transient HV interval block followed by recovery from the block at shorter coupling intervals without prolongation of the H 1H 2 and H 2V 2 intervals, suggesting true supernormal conduction. Isoproterenol enhanced the supernormal conduction, with shortening of blocked intervals and recovery of atrioventricular conduction, while atropine caused their less marked enhancement. Linking (ie, retrograde concealment of the impulse to the distal His bundle region through the blocked right bundle branch) is considered a possible mechanism of supernormal conduction in this case.

Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli.

Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.

Hydrogen bond network in the distal site of peroxidases: spectroscopic properties of Asn70 --> Asp horseradish peroxidase mutant.

The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, 1H NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the 1H NMR signal of heme peripheral 8-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.

Catalytic activities and structural properties of horseradish peroxidase distal His42 --> Glu or Gln mutant.

The distal histidine (His) is highly conserved in peroxidases and has been considered to play a major role as a general acid-base catalyst for peroxidase reaction cycle. Recently, however, the X-ray structure of chloroperoxidase from the marine fungus Caldariomyces fumago has revealed that a glutamic acid is located at the position where most of the peroxidase has a histidine residue, suggesting that the carboxyl group in the glutamic acid (Glu) can also assist cleavage of an O-O bond in peroxides [Sundaramoorthy, M., Terner, J., & Poulos, T. L. (1995) Structure 3, 1367-1377]. In order to investigate catalytic roles of the glutamic acid at the distal cavity, two horseradish peroxidase mutants were prepared, in which the distal His42 has been replaced by Glu (H42E) or Gln (H42Q). The formation rate of compound I in the H42E mutant was significantly greater than that for the H42Q mutant, indicating that the distal Glu can play a role as a general acid-base catalyst. However, the peroxidase activity of the H42E mutant was still lower, compared to that for native enzyme. On the basis of the CD, resonance Raman, and EPR spectra, it was suggested that the basicity of the distal Glu is lower than that of the distal His and the position of the distal Glu is not fixed at the optimal position as a catalytic amino acid residue, although no prominent structural changes around heme environment were detected. The less basicity and improper positioning of the distal Glu would destabilize the heme-H2O2-distal Glu ternary intermediate for the peroxidase reaction. Another characteristic feature in the mutants was the enhancement of the peroxygenase activity. Since the peroxygenase activity was remarkably enhanced in the H42E mutant, the distal Glu is also crucial to facilitate the peroxygenase activity as well as the enlarged distal cavity caused by the amino acid substitution. These observations indicate that the distal amino acid residue is essential for function of peroxidases and subtle conformational changes around the distal cavity would control the catalytic reactions in peroxidase.

Fluoride binding in hemoproteins: the importance of the distal cavity structure.

The electronic absorption and resonance Raman spectra of the fluoride complexes of various peroxidases and selected site-directed mutants have been studied at pH 5.0, and compared to the spectra obtained for the myoglobin-F adduct. It is shown that the electronic absorption maxima depend on the degree of conjugation between the porphyrin macrocycle and the vinyl substituents. Moreover, it is confirmed that the wavelength of the CT1 band is a sensitive probe of axial ligand polarity and of its interaction with the distal protein residues. The results highlight the different mechanism of stabilization of the fluoride ligand exerted by the distal residues in myoglobin and peroxidases. In peroxidases, the Arg is determinant in controlling the ligand binding via a strong hydrogen bond between the positively charged guanidinium group and the anion. Mutation of Arg to Leu decreases the stability of the complex by 900-fold, suggesting that this interaction stabilizes the complex by 4 kcal/mol. The distal His also contributes to the stability of the fluoride complex, presumably by accepting a proton from HF and hydrogen-bonding, through a water molecule, to the anion. Mutation of His to Leu decreases the stability of the fluoride complex by 30-fold, suggesting that this interaction is much weaker than the interaction with the distal Arg. For Mb, the distal His is solely responsible for stabilization of the exogenous ligand.

Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(III) complex

A complex of an independent L chain from anti-meso-tetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90°C). Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment. The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone. Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115–146) and hydrophilic region (147–189) in C l are important for thermostability and activity, respectively. Tertiary structure of L-zyme was predicted by AbM. Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity. Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively. Distance between proximal His and distal His in L-zyme is 9.09 Å, whereas that of ARP is 7.8 Å.

Triangular kinetic schemes applied to the stability of a Heme—Globin complex

Horse heart apomyglobin traps the heme released from Aplysia california myoglobin. The kinetics fit a triangular mechanism for a biphasic reaction. Laplacian solutions for differential equations appropriate to triangular kinetic schemes involving up to four rate constants are elaborated and confirmed. Two general schemes and two special case are considered. In the first scheme, a rearrangement of the starting material is concurrent with product formation. In the second scheme, the starting material forms two products in equilibrium at two different rates. A general equation for the absorbance-time curve is derived for these triangular schemes, from which rate constants can be estimated. Changes in instantaneous rates versus time are employed to analyze the absorption versus time plots and the curvature of a first-order rate analysis. Aplysia metmyglobin equilibrates between slow donor (pentacoordinate, which lacks the axial water molecule) and fast donor (hexacoordinate). No heme release was observed for deoxy, oxy, carbonyl, or azide derivatives of the Aplysia myoglobin, or when the distal His E7 of the apohemoprotein is replaced by leucine or valine. This suggests a role for hydrophobicity of the active site, and for a trans effect of the axial ligand in determining the stability of the embedded prosthethic heme.

Site-directed mutations at phenylalanine-190 of manganese peroxidase: effects on stability, function, and coordination.

A series of site-directed mutants, F190Y, F190L, F190I, and F190A, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium was generated by overlap extension with the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The manganese peroxidase variants (MnPs) were purified and characterized by kinetic and spectroscopic methods. At pH 4.5, the UV-vis spectra of the ferric and oxidized states of the mutant proteins were very similar to those of the wild-type enzyme. Steady-state kinetic analyses showed that the apparent Km and k(cat) values for MnII and H2O2 also were similar to the corresponding values for the wild-type MnP. The apparent Km and k(cat) values for ferrocyanide oxidation by MnP were not affected by the F190Y, F190L, or F190I mutations; however, the apparent Km value for ferrocyanide oxidation by the F190A mutant MnP was approximately 1/8 of that for the wild-type enzyme. Likewise, the apparent k(cat) value for ferrocyanide oxidation by the MnP F190A mutant was approximately 4-fold greater than the corresponding k(cat) for the wild-type MnP. The stabilities of both the native and oxidized states of MnP were significantly affected by several of the mutations at Phe190. Replacement of Phe190 by either Ile or Ala significantly destabilized the resultant proteins to thermal denaturation. Moreover, the rates of spontaneous reduction of the oxidized intermediates, MnP compounds I and II, were dramatically increased for the F190A mutant relative to the rates observed for the wild-type enzyme. The spectroscopic properties of the wild-type and F190 mutant MnPs were examined as a function of pH. At room temperature, increasing pH from 5.0 to 8.5 induced a FeIII high- to low-spin transition for all of the MnP proteins. This transition may involve direct coordination of the distal His residue to the heme iron to produce bishistidinyl coordination as suggested by magnetic circular dichroism spectroscopy. The pH at which this transition occurred was considerably lower for the F190A and F190I variants and suggests that Phe190 plays a critical role in stabilizing the heme environment of MnP.

Molecular Dynamics Simulation Study of the B-States of Solvated Carbon Monoxymyoglobin

Classical molecular dynamics simulation is employed to study the photodissociated B-states of carbon monoxymyoglobin. Four independent 10 ps dynamical trajectories of the fully solvated protein, in a box of 2988 water molecules using periodic boundary conditions, were simulated at 300 K starting from the equilibrium deoxymyoglobin structure. Two prototypical protein conformational substates, defined by the conformation of the distal histidine, were sampled from a 1.5 ns trajectory of solvated, room temperature deoxymyoglobin. The structure and ligand vibrational spectral line shapes and frequency shifts were derived from the simulation trajectories. The simulated structure of the ligand−heme complex shows that the ligand is localized under the C-ring of the heme, which is consistent with the low-temperature X-ray crystal structures of the photolyzed state [Schlichting et al. Nature 1994, 371, 808. Teng; Šrajer; Moffat Nat. Struct. Biol. 1994, 1, 701]. The ligand is found to be parallel to the heme “plane” and confined to a small region displaced from the heme iron in apparent agreement with the low-temperature X-ray crystal structures and room temperature experimental studies [Lim; Jackson; Anfinrud Science 1995, 269, 962]. Stark shifts in the ligand vibrational frequency, induced by the protein electric field in the heme pocket, are estimated using both atomic point charge models with explicit solvent and finite difference Poisson−Boltzmann calculations. Simulated ligand frequency shifts are compared with those of the experimental spectrum as a test of the accuracy of the simulated electric field in the heme pocket. The magnitudes of the induced ligand frequency shifts are in good agreement with experimental results. The largest contribution to the electric field in the heme pocket is due to the heme and proximal histidines not, as it has often been assumed, the distal histidine. The exact value of the protein electric field experienced by the ligand is well correlated with the conformation of the distal His. The dipole induced in the ligand by the protein electric field is found to make a significant contribution to the total ligand dipole moment and is not necessarily aligned with the ligand bond. The proposal that the ligand “docking site” functions to suppress CO binding is examined.

The Distal Glutamic Acid as an Acid-Base Catalyst in the Distal Site of Horseradish Peroxidase

The distal His is an essential amino acid residue as a general acid-base catalyst for peroxidase reaction cycle. However, the x-ray structure of chloroperoxidase revealed that Glu is located near the heme, suggesting that the carboxyl group also assists cleavage of O-O bond in peroxides. In this paper, we examined functional and structural properties of a horseradish peroxidase mutant having Glu instead of the distal His. Although this amino acid replacement depressed reaction rate with H2O2and oxidation activity for guaiacol, the mutant still exhibited much higher activity than mutants in which the distal His was replaced by hydrophobic amino acid. Kinetic measurements suggest that the proton abstraction is decelerated in the mutant due to large fluctuation of the carboxyl group of the distal Glu. Therefore, we can conclude that Glu can be a potent acid-base catalyst for peroxidase reaction cycle, if the carboxyl group can be fixed at the optimum position.

1H NMR Study of the Heme Molecular Structure in Sperm Whale Met-Aquo and Met-Imidazole Myoglobins

Abstract The resolved heme peripheral side-chain proton resonances of sperm whale met-aquo myoglobin have been connected to the corresponding resonances in met-imidazole myoglobin by the magnetization transfer through the external ligand exchange. From the signal assignments in met-imidazole myoglobin, which have been independently obtained using conventional two-dimensional NMR and one-dimensional nuclear Overhauser effect difference spectroscopies, the observed connectivities unambiguously identified the corresponding resonances of met-aquo myoglobin. The calculation of the pseudo-contact shifts for the resonances of met-aquo myoglobin from the reported zero-field splitting constant [Y. -H. Kao, and J. T. J. Lecomte, J. Am. Chem. Soc., 115, 9754—9762 (1993) and J. Am. Chem. Soc., 116, 6991 (1994)] together with the diamagnetic shifts observed for carbonmonoxy myoglobin [B. C. Mabbutt, and P. E. Wright, Biochim. Biophys. Acta, 832, 175—185 (1985)], allowed the extraction of the contact shifts from their paramagnetic shifts. The heme peripheral propionate conformation in met-aquo myoglobin has been determined from the analysis of the contact shifts for the CαH2 proton resonances. The obtained conformations agreed well with those described by the X-ray study [T. Takano, J. Mol. Biol., 110, 537—568 (1977)]. The thermal spin equilibrium in met-imidazole myoglobin has been analyzed using the mean heme methyl proton shift as an indicator for the spin state of the protein. The equilibrium populations determined from the analysis of the NMR shift data did not agree with those determined from the magnetic susceptibility measurements [T. Iizuka, and M. Kotani, Biochim. Biophys. Acta,181, 275—286 (1969)], indicating that the heme methyl proton shifts cannot be used as quantitative probe for the thermal spin equilibrium in met-imidazole myoglobin. This would be attributed to the temperature-dependent delocalization of the unpaired electron density from the heme iron to the porphyrin π system and the axial ligands due to the perturbation on the coordination structure around the iron, exerted by the steric repulsion between the bound imidazole and the imidazole side-chain of the distal His residue, as revealed by the X-ray study [C. Lionetti, M. G. Guanziroli, F. Frigerio, P. Ascenzi, and M. Bolognesi, J. Mol. Biol., 217, 409—412 (1991)].

Aplysia limacina myoglobin cDNA cloning: an alternative mechanism of oxygen stabilization as studied by active-site mutagenesis.

The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back transplanting the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor delta-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s(-1) [as compared with the value of 15 s(-1) for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A-H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s(-1) to more than 700 s(-1), in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)-containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved.

Catalytic roles of the distal site asparagine-histidine couple in peroxidases.

There are highly conserved hydrogen bonds between the distal His and the adjacent Asn in many peroxidases. Although the crystal structure of horseradish peroxidase C (HRP) is not available, comparison of the amino acid sequence with cytochrome c peroxidase indicates that Asn70 is making the hydrogen bond with the distal His in the active site of HRP. To investigate the catalytic roles of the hydrogen bond, Asn70 in HRP was replaced with Val (N70V) or Asp (N70D). Though UV-vis, CD, and 1H-NMR spectra of native (plant enzyme), wild-type (recombinant enzyme), and mutant HRPs suggest that the active site and secondary structure are very similar even after mutation, the mutants exhibit low Vmax values for the hydroquinone oxidation (native, 281; wild-type, 283; and N70V, 18; and N70D, 33 microM.min-1). The rates of compound I formation were decreased to less than 10% of that of the native enzyme. The reduction rates of compounds I and II by guaiacol also were reduced to less than 10% of that of the native enzyme. Substituent effects of various phenol derivatives on the reduction of native, wild-type, and mutant compound I were examined. Large negative Hammett rho values (rho N70V:fast = -4.0, rho N70V:slow = -3.6, rho N70D = -3.8, rho native = -6.9, and rho wild-type = -6.8) are an indication of electron transfer being the rate-determining step in the phenol oxidation. However, these results also indicate the participation of the deprotonation step in the compound I reduction process. The proton abstraction from phenol must be harder for the mutants due to the decrease of basicity of the distal His upon mutation. Contrary to phenol oxidation, ABTS [2,2'-azinobis(3-ethybenzothiazoline-6-sulfonic acid)] oxidation activity was substantially increased by the mutations (native, 73; wild-type, 71; N70V, 217; and N70D, 234 microM.s-1). The redox potentials of N70V and N70D compounds II are 957 and 970 mV (vs NHE), which are 95 and 108 mV higher than that of native compound II (862 mV), respectively. Therefore, the high ABTS oxidation activities of mutants are attributed to these high redox potentials of compound II.