NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR evidence for a structural alteration on the active site of G. japonicus myoglobin upon azide ion binding

The heme molecular structure of the met-azido form of the myoglobin from the shark Galeorhinus japonicus has been investigated by 1H NMR. A nuclear Overhauser effect (NOE) was clearly observed among the heme peripheral side-chain proton signals of this complex, which undergoes thermal spin equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states, and the NOE connectivities provided the assignment of the resonances from the heme C13(1)H2 and C17(1)H2 protons. Chemical shift inequivalence of these proton resonances not only provided information about the orientation of these methylene protons with respect to the heme plane, but also allowed characterization of the time-dependent build-up of the NOE between them, which yields the correlation time for the internal motion of the inter-proton vector. The relatively large mobility found for the C17(1)H2 group suggests that the carboxyl oxygen of the heme C17 propionate is not anchored to the apo-protein by a salt bridge. It has been shown that the ferric high-spin form of G. japonicus Mb possesses a penta-coordinated heme [Suzuki, T. (1987) Biochim. Biophys. Acta 914, 170-176; Yamamoto, Y., Osawa, A., Inoue, Y., Ch?j?, R. & Suzuki, T. (1990) Eur. J. Biochem. 192, 225-229] and that the conformation of both heme propionate groups is fixed with respect to the heme, as well as the apo-protein, by a salt bridge [Yamamoto, Y., Inoue, Y., Ch?j?, R. & Suzuki, T. (1990) Eur. J. Biochem. 189, 567-573]. Therefore the weakening or interruption of the interaction between the C17 propionate and His FG3 upon the changes of the coordination and spin state of the heme iron, during azide ion binding to ferric high-spin G. japonicus Mb, is attributed to the displacement of the FG corner of the apoprotein away from the heme C17 propionate group. A similar structural alteration has been revealed by X-ray structural analyses of unliganded and liganded forms of ferrous hemoproteins [Baldwin, J. & Chothia, C. (1979) J. Mol. Biol. 129, 175-220; Phillips, S.E.V. (1980) J. Mol. Biol. 142, 531-554].     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].     

Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F., & Brunori, M. (1987) Biochem. J. 246, 787-789].     

Spectra of intermediates in oxidation and reduction of cytochrome c oxidase

Two kinetic components with distinct difference spectra occur during reduction of cytochrome c oxidase by ruthenium hexamine. They are attributed to reduction of heme a (fast phase) and heme a3 (slow phase) (Scott, R. A., and Gray, H. B. (1980) J. Am. Chem. Soc. 102, 3219-3774). Two spectra seen during oxidation of cytochrome c oxidase by molecular oxygen have also been attributed to oxidation of hemes a3 and a (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787). We now report that spectra for the reductive and oxidative reactions obtained with the same preparations and the same apparatus under similar conditions are significantly different. The reactions appear to populate different reaction intermediates. Reconstitution into phospholipid vesicles does not affect these two spectra significantly. During turnover, the chief intermediates are those of the reductive pathway (Scott and Gray type intermediates). Reduction of heme a3 occurs approximately 70 times faster after turnover than the reduction of the resting enzyme. This is probably a dramatic "pulsing" effect (Wilson, M. T., Peterson, J., Antonini, E., Brunori, M., Colosimo, A., and Wyman, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 7115-7118).     

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.     

Assignment of heme methyl 1H-NMR resonances of high-spin and low-spin ferric complexes of cytochrome p450cam using one-dimensional and two-dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments.

An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.     

Kinetics of cyanide binding to Chromatium vinosum ferricytochrome c'

The kinetics of the reversible binding of cyanide by the ferric cytochrome c' from Chromatium vinosum have been studied over the pH range 6.9-9.6. The reaction is extremely slow at neutral pH compared to the reactions of other high-spin ferric heme proteins with cyanide. The observed bimolecular rate constant at pH 7.0 is 2.25 X 10(-3) M-1 s-1, which is approximately 10(7)-fold slower than that for peroxidases, approximately 10(5)-fold slower than those for hemoglobin and myoglobin, and approximately 10(2)-fold to approximately 10(3)-fold slower than that recently reported for the Glycera dibranchiata hemoglobin, which has anomalously slow cyanide rate constants of 4.91 X 10(-1), 3.02 X 10(-1), and 1.82 M-1 s-1 for components II, III, and IV, respectively [Mintorovitch, J., & Satterlee, J. D. (1988) Biochemistry 27, 8045-8050; Mintorovitch, J., Van Pelt, D., & Satterlee, J. D. (1989) Biochemistry 28, 6099-6104]. The unusual ligand binding property of this cytochrome c' is proposed to be associated with a severely hindered heme coordination site. Cyanide binding is also characterized by a nonlinear cyanide concentration dependence of the observed rate constant at higher pH values, which is interpreted as involving a change in the rate-determining step associated with the formation of an intermediate complex between the cytochrome c' and cyanide prior to coordination. The pH dependence of both the binding constant for the formation of the intermediate complex and the association rate constant for the subsequent coordination to the heme can be attributed to the ionization of HCN, where cyanide ion binding is the predominant process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium and kinetics of the reaction of Aplysia myoglobin with azide.

The present paper reports a study on the equilibria and kinetics of the acid-alkaline transition and the azide binding reaction by ferric Aplysia myoglobin. A single completely reversible spectrophotometric titration curve is found over the pH range from similar to 5 to similar to 9, with an apparent pK equals to 7.5 for the acid-alkaline transition. The kinetics of the process, followed by the temperature-jump method, gives, at pH values close to the pK of the transition, one single, well-resolved, relaxation independent of protein concentration and of type of buffer used. The pattern accords to a simple pH dependent reaction, in buffered medium, between the two forms of the protein. The results of the azide binding reaction show that the process conforms to simple equilibrium as expected for a single site protein. The méasured association constant is reported as a function of pH. The kinetics of the reaction of Aplysia metMb with N3- minus shows, on the other hand, a complex behavior. The relaxation pattern is found to strongly depend on pH and ligand concentration in such a way to suggest a linkage between ligand binding and acid-alkaline transition. The system is discussed on the basis of two simplifying conditions, i.e., at low and higher pH with respect to the pK of the acid-alkaline transition. At acid pH the reaction corresponds to a single bimolecular process as expected for a simple binding reaction; at alkaline pH, the dependence of relaxation time on ligand concentration implies the existence of a rate-limiting monomolecular step. On the basis of a reaction scheme implying that binding of the ligand can only occur through the acid (aquomet) form of the protein via the displacement of the water molecule, the experimental data are quantitatively accounted for.     

Control of cytochrome oxidase activity. A transient spectroscopy study.

The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.     

Does the reduction of c heme trigger the conformational change of crystalline nitrite reductase?

The structures of nitrite reductase from Paracoccus denitrificans GB17 (NiR-Pd) and Pseudomonas aeruginosa (NiR-Pa) have been described for the oxidized and reduced state (Fül?p, V., Moir, J. W. B., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Nurizzo, D., Silvestrini, M. C., Mathieu, M., Cutruzzolà, F., Bourgeois, D., Fül?p, V., Hajdu, J., Brunori, M., Tegoni, M., and Cambillau, C. (1997) Structure 5, 1157-1171; Nurizzo, D., Cutruzzolà, F., Arese, M., Bourgeois, D., Brunori, M., Cambillau, C. , and Tegoni, M. (1998) Biochemistry 37, 13987-13996). Major conformational rearrangements are observed in the extreme states although they are more substantial in NiR-Pd. The four structures differ significantly in the c heme domains. Upon reduction, a His17/Met106 heme-ligand switch is observed in NiR-Pd together with concerted movements of the Tyr in the distal site of the d1 heme (Tyr10 in NiR-Pa, Tyr25 in NiR-Pd) and of a loop of the c heme domain (56-62 in NiR-Pa, 99-116 in NiR-Pd). Whether the reduction of the c heme, which undergoes the major rearrangements, is the trigger of these movements is the question addressed by our study. This conformational reorganization is not observed in the partially reduced species, in which the c heme is partially or largely (15-90%) reduced but the d1 heme is still oxidized. These results suggest that the d1 heme reduction is likely to be responsible of the movements. We speculate about the mechanistic explanation as to why the opening of the d1 heme distal pocket only occurs upon electron transfer to the d1 heme itself, to allow binding of the physiological substrate NO2- exclusively to the reduced metal center.     

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 A resolution

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.19. The tertiary structure of the molecule conforms to the common globin fold, consisting of eight alpha-helices. The N-terminal helix A and helix G deviate significantly from linearity. The distal residue is recognized as Val63 (E7), which, however, does not contact the heme directly. Moreover the sixth (distal) co-ordination position of heme iron is not occupied by a water molecule at neutrality, i.e. below the acid-alkaline transition point of A. limacina myoglobin. The heme group sits in its crevice in the conventional orientation and no signs of heme isomerism are evident. The iron atom is 0.26 A out of the porphyrin plane, with a mean Fe-N (porphyrin) distance of 2.01 A. The co-ordination bond to the proximal histidine has a length of 2.05 A, and forms an angle of 4 degrees with the heme normal. A plane containing the imidazole ring of the proximal His intersects the heme at an angle of 29 degrees with the (porphyrin) 4N-2N direction. Inspection of the structure of pH 9.0 indicates that a hydroxyl ion is bound to the Fe sixth co-ordination position.     

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.     

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis

The 2.2-A X-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli [Fishel, L.A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360], has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53----Ile substitution in CCP(MI). A Trp-51----Phe mutant remains pentacoordinated and exhibits only minor distal structural adjustments. The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron [Smulevich, G., Mauro, J.M., Fishel, L. A., English, A. M., Kraut, J., & Spiro, T. G. (1988) Biochemistry 27, 5477-5485]. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191----Phe mutant that has substantially diminished enzyme activity and altered magnetic properties [Mauro, J. M., Fishel, L. F., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256] accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large (ca. 1 A) local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structure rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. Changing proximal Asp-235 to Asn results in two significant localized structural changes. First, the heme iron moves toward the porphyrin plane, and distal water 595 now clearly resides in the iron coordination sphere at a distance of 2.0 A. The observation of hexacoordinated iron for the D235N mutant is in accord with previous resonance Raman results. Second, the indole side chain of Trp-191 has flipped over as a result of the mutation; the tryptophan N epsilon takes part in a new hydrogen bond with the backbone carbonyl oxygen of Leu-177.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature jump relaxation kinetics of the P-450cam spin equilibrium

The ferric spin-state equilibrium and relaxation rate of cytochrome P-450 has been examined with temperature jump spectroscopy using a number of camphor analogues known to induce different mixed spin states in the substrate-bound complexes [Gould, P., Gelb, M., & Sligar, S. G. (1981) J. Biol. Chem. 256, 6686]. All temperature-induced spectral changes were monophasic, and the spin-state relaxation rate reached a limiting value at high substrate concentrations. The ferric spin equilibrium constant, Kspin, is defined in terms of the rate constants k1 and k-1 via Kspin = k1/k-1 = [P-450(HS)]/[P-450(LS)] where HS and LS represent high-spin (S = 5/2) and low-spin (S = 1/2) ferric iron, respectively, and the spectrally observed spin-state relaxation rate by kobsd = k1 + k-1. A strong correlation between the fraction of high-spin species and the rate constant, k-1, is observed. For a 3 degrees C temperature jump (from 10 to 13 degrees C), the 23% high-spin tetramethylcyclohexanone complex (Kd = 45 +/- 20 microM) is characterized by a ferric spin relaxation rate of kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and camphoroquinone (75% high spin, Kd = 15 +/- 5 microM) complexes are 1430 and 346 s-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy.

Proton NMR spectroscopy was applied to myoglobin in the ferric, water-liganded form (metMbH2O) and the apo form (apoMb) to probe the structure and stability of the latter. Proteins from sperm whale and horse skeletal muscles were studied to simplify the spectral assignment task. Nuclear Overhauser effects and the response of chemical shifts to variations of pH were used as indicators of residual native holoprotein structure in the apoprotein. The investigation was focused in the histidine side chains and their environment. In metMbH2O, the resonances of all imidazole rings not interacting with the heme were assigned by applying standard two-dimensional methods. These assignments were found to differ from those reported elsewhere [Carver, J. A., & Bradbury, J. H. (1984) Biochemistry 23, 4890-4905] except for His-12, -113, and -116. Only one histidine (His-36) has a pK(a) higher than 7, two (His-48 and His-113) have a pK(a) lower than 5.5, and two (His-24 and His-82) appear not to titrate between pH 5.5 and pH 10. In the apoproteins, the signals of His-113 and His-116, as well as those of His-24, -36, -48, and -119 previously assigned in the horse globin [Cocco, M. J.. & Lecomte, J. T. J. (1990) Biochemistry 29, 11067-11072], could be followed between pH 5 and pH 10. A comparison to the holoprotein data indicated that heme removal has limited effect on the pK(a) and the surroundings of these residues. Five additional histidines which occur in the two helices and connecting loops forming the heme binding site were identified in the horse apoprotein. Four of these were found to have pK(a) values lower than that expected of an exposed residue. The NOE and titration data were proposed to reflect the fact that several holoprotein structural elements, in particular outside the heme binding site, are maintained in the apoprotein. In the heme binding region of the apoprotein structure, the low pK(a)'s suggest local environments which are resistant to protonation.     

Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.