Comparison of the data, analysis, and results of X-ray absorption studies of cytochrome c oxidase

Differences in the methods of analysis of X-ray absorption data used by Powers et al. [Powers, L., Blumberg, W. E., Chance, B., Barlow, C., Leigh, J., Jr., Smith, J., Yonetani, T., Vik, S., & Peisach, J. (1979) Biochim. Biophys. Acta 547, 520-538; Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] and Scott et al. [Scott, R., Schwartz, J., & Cramer S. (1986) Biochemistry 25, 5546-5555] are clarified. In addition, we compare the X-ray absorption data and results for resting cytochrome c oxidase reported by both groups using the same analysis method and conclude apart from any assumptions that the data are not identical.     

Kinetic, structural, and spectroscopic identification of geminate states of myoglobin: a ligand binding site on the reaction pathway

Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.     

Cytochrome c peroxidase compound ES is identical with horseradish peroxide compound I in iron-ligand distances

X-ray absorption studies of compound ES of cytochrome c peroxidase show a short iron-oxygen distance of 1.67 +/- 0.04 A, an iron-histamine distance of 1.91 +/- 0.03 A, and an iron-pyrrole nitrogen average distance of 2.02 +/- 0.02 A. This is identical within the error with the reported structure of horseradish peroxidase compound I [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611]. Comparisons of the structures of myoglobin peroxide [Chance, M., Powers, L., Kumar, C., & Chance, B. (1986) Biochemistry (preceding paper in this issue)], compound ES, and the intermediates of horseradish peroxidase reveal the possible mechanisms for the stabilization of the free radical species generated during catalysis. The proximal histidine regulates the structure and function of the pyrrole nitrogens and the heme, allowing for the formation and maintenance of the characteristic intermediates.     

A spectral study of cobalt(II)-substituted Bacillus cereus phospholipase C

The coordination sphere of both the structural and catalytic zinc ions of Bacillus cereus phospholipase C has been probed by substitution of cobalt(II) for zinc and investigation of the resultant derivatives by a variety of spectroscopic techniques. The electronic absorption, circular dichroic, magnetic circular dichroic, and electron paramagnetic resonance spectra were found to be strikingly similar when cobalt(II) was substituted into either site and are consistent with a distorted octahedral environment for the metal ion in both sites. Octahedral coordination appears comparatively rare in zinc metalloenzymes but has been suggested for glyoxalase I [Sellin, S., Eriksson, L. E. G., Aronsson, A.-C., & Mannervik, B. (1983) J. Biol. Chem. 258, 2091-2093; Garcia-Iniguez, L., Powers, L., Chance, B., Sellin, S., Mannervik, B., & Mildvan, A. S. (1984) Biochemistry 23, 685-689], transcarboxylase [Fung, C.-H., Mildvan, A. S., & Leigh, J. S. (1974) Biochemistry 13, 1160-1169], and the regulatory binding site of Aeromonas aminopeptidase [Prescott, J. M., Wagner, F. W., Holmquist, B., & Vallee, B. L. (1985) Biochemistry 24, 5350-5356]. Phospholipase C is so far unique in having two such sites.     

CO bond angle changes in photolysis of carboxymyoglobin

Previous studies [Chance, B., Fischetti, B., & Powers, L. (1983) Biochemistry 22, 3820-3829] of the local structure changes around the iron in carboxymyoglobin on photolysis at 4 K revealed that the iron-carbon distance increased approximately 0.05 A but was accompanied by a lengthening of the iron-pyrrole nitrogen bonds of the heme (approximately 0.03 A) that was not as large as that found in the deoxy form. Further analysis of these data together with comparison to model compounds indicates that the Fe-C-O bond angle in carboxymyoglobin is bent (127 +/- 4 degrees), having a structure identical, within the error, with the "pocket" porphyrin model compound FePocPiv(1-MeIm)(CO) [Collman, J. P., Brauman, J. I., Collins, T. J., Iverson, B. L., Lang, G., Pettman, R., Sessler, J. L., & Walters, M. A. (1983) J. Am. Chem Soc. 105, 3038-3052]. On photolysis, this angle decreases by 5-10 degrees. In addition, correlation is observed between the increase in the length of the Fe-C bond and the decrease of the Fe-C-O angle. These results suggest that the rate-limiting step in recombination is the thermal motion of CO in the pocket to achieve an appropriate bonding angle with respect to the iron. These changes constitute the first molecular picture of the photolysis process, as well as the structure of the geminate state, and are important in clarifying nuclear tunneling parameters.     

Structures of photolyzed carboxymyoglobin

The structures of photoactivated carboxymyoglobin (Mb*CO) at temperatures to 10 K have been investigated by Fourier transform infrared (FT-IR) spectroscopy, visible spectroscopy, and near-infrared spectroscopy. Two energy states for *CO are observed by FT-IR, which are altered in frequency by 94% and 88% of the difference from the ground-state heme CO toward free CO gas [Alben, J. O., Beece, D., Bowne, S. F., Doster, W., Eisenstein, L. Frauenfelder, H., Good, D., McDonald, J. D., Marden, M. C., Moh, P. P., Reinisch, L., Reynolds, A. H., Shyamsundar, E., & Yue, K. T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3744-3748]. Ground-state MbCO shows no absorption in the near-infrared from 700 to 1200 nm. Conversely, Mb*CO shows an absorption near 766 nm, similar to that of ferrous myoglobin (deoxy-Mb) at 758 nm. These data are compared with M?ssbauer isomer shifts and quadrupole splitting [Spartalian, K., Lang, G., & Yonetani, T. (1976) Biochim. Biophys. Acta 428, 281-290] and magnetic susceptibility measurements [Roder, H., Berendzen, J., Bowne, S. F., Frauenfelder, H., Sauke, T. B., Shyamsunder, E., & Weissman, M. B. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2359-2363], which clearly indicate that the iron in both Mb*CO and deoxy-Mb is in the high-spin Fe(II) state, as does the heme transition in the Soret [Iizuka, T., Yamamoto, H., Kotani, M., & Yonetani, T. (1974) Biochim. Biophys. Acta 371, 126-139]. Thus the electronic structure of iron in Mb*CO is nearly identical with that of deoxy-Mb, and *CO is only slightly perturbed from the free gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural aspects of the copper sites in cytochrome c oxidase. An X-ray absorption spectroscopic investigation of the resting-state enzyme

Copper K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the structural details of the coordination environment of the copper sites in eight resting-state samples of beef heart cytochrome c oxidase prepared by different methods. The unusual position and structure of the resting-state copper edge spectrum can be adequately explained by the presence of sulfur-containing ligands, with a significant amount of S----Cu(II) charge transfer (i.e., a covalent site). Quantitative curve-fitting analysis of the copper extended X-ray absorption fine structure (EXAFS) data indicates similar average first coordination spheres for all resting-state samples, regardless of preparation method. The average coordination sphere (per 2 coppers) mainly consists of 6 +/- 1 nitrogens or oxygens at an average Cu-(N,O) distance of 1.99 +/- 0.03 A and 2 +/- 1 sulfurs at an average Cu-S distance of 2.28 +/- 0.02 A. Quantitative curve-fitting analysis of the outer shell of the copper EXAFS indicates the presence of a Cu...Fe interaction at a distance of 3.00 +/- 0.03 A. Proposed structures of the two copper sites based on these and other spectroscopic results are presented, and differences between our results and those of other published copper XAS studies [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] are discussed.     

X-ray absorption studies of myoglobin peroxide reveal functional differences between globins and heme enzymes

X-ray absorption studies of myoglobin peroxide show that although it is not identical with compound I or II of horseradish peroxidase [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611], it has some structural features in common with both. As seen in compound I, the Fe-O distance is short, but the iron-pyrrole nitrogen distance is contracted with a longer iron-histidine distance like compound II. The iron has a higher oxidation state than Fe3+, suggesting an oxyferryl ion type species. Comparison of the structures of various peroxidase and myoglobin compounds points out systematic differences that may explain the catalytic activity of the pi cation radical as well as some of the differences between globins and heme enzymes.     

Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding

The extended X-ray absorption fine structure (EXAFS) data show differences between the active site structures of different cytochrome oxidase preparations. In the resting (as isolated) state of the Yonetani preparation, the bridging atom between Fe3+a3 and Cu2+a3 is present [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465], whereas in another preparation (e.g., Hartzell-Beinert), this atom seems to be bound only to Fe3+a3 in a significant fraction of the molecules. Both preparations bind cyanide in a multiphasic fashion, suggesting that the resting cytochrome oxidase is not homogeneous but rather is a mixture of several forms. The proportion of these forms as detected by cyanide binding kinetics differs for different preparations. However, upon reduction and reoxidation (conversion to the "oxygenated" form) the cyanide binding kinetics become monophasic and all preparations of the oxygenated form bind cyanide at the same rate. Thus, a combination of structural and kinetic approaches seems necessary for evaluation of the nature of the active site of cytochrome oxidase in its various forms.     

A large reservoir of sulfate and sulfonate resides within plasma cells from Ascidia ceratodes, revealed by X-ray absorption near-edge structure spectroscopy

The study of sulfur within the plasma cells of Ascidia ceratodes [Carlson, R. M. K. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2217-2221; Frank, P., Carlson, R. M. K., & Hodgson, K. O. (1986) Inorg. Chem. 25, 470-478; Hedman, B., Frank, P., Penner-Hahn, J. E., Roe, A. L., Hodgson, K. O., Carlson, R. M. K., Brown, G., Cerino, J., Hettel, R., Troxel, T., Winick, H., & Yang, J. (1986) Nucl. Instrum. Methods Phys. Res., Sect. A 246, 797-800] has been extended with X-ray absorption near-edge structure (XANES) spectroscopy. An intense absorption feature at 2482.4 eV and a second feature at 2473.7 eV indicate a large endogenous sulfate concentration, as well as smaller though significant amounts of thiol or thioether sulfur, respectively. A strong shoulder was observed at 2481.7 eV on the low-energy side of the sulfate absorption edge, deriving from a novel type of sulfur having a slightly lower oxidation state than sulfate sulfur. The line width of the primary transition on the sulfur edge of a vanadium (III) sulfate solution was found to be broadened relative to that of sodium sulfate, possibly deriving from the formation of the VSO4+ complex ion [Britton, H. T. S., & Welford, G. (1940) J. Chem. Soc., 761-764; Duffy, J. A., & Macdonald, W. J. D. (1970) J. Chem. Soc., 977-980; Kimura, T., Morinaga, M., & Nakano, J. (1972) Nippon Kagaku Zaishi, 664-667]. Similar broadening appears to characterize the oxidized sulfur types in vanadocytes. A very good linear correlation between oxidation state and peak position (in electronvolts) was found for a series of related sulfur compounds. This correlation was used to determine a 5+ oxidation state for the additional sulfur type at 2481.7 eV. (ABSTRACT TRUNCATED AT 250 WORDS)     

Extended X-ray absorption fine structure of copper in CuA-depleted, p-(hydroxymercuri)benzoate-modified, and native cytochrome c oxidase

Cytochrome c oxidase contains four redox-active metal centers: two heme irons, cytochromes a and a3, and two copper ions, CuA and CuB. Due to the paucity of spectroscopic signatures for both copper sites in cytochrome c oxidase, the ligands and structures for these sites have remained ambiguous. The specific depletion of CuA from the p-(hydroxymercuri)benzoate- (pHMB-) modified cytochrome c oxidase recently reported [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972] is herein described. Characterization of this enzyme shows that the structures of the remaining metal centers are essentially unperturbed by the CuA modification and depletion (P. M. Li, J. Gelles, and S. I. Chan, unpublished results). Copper extended X-ray absorption fine structure (EXAFS) measurements on the CuA-depleted cytochrome c oxidase reveal coordination of three (N, O) ligands and one (S, Cl) ligand at the CuB site. Comparison of EXAFS results obtained for the CuA-depleted, pHMB-modified, and "unmodified control" enzymes has allowed the deconvolution of the EXAFS in terms of the inner coordination spheres for CuA as well as CuB. On the basis of these data, it is found that the structure for the CuA site is consistent with two (N, O) ligands and two S ligands.     

X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for Fe-S interaction in the enzyme-substrate complex.

Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

Description of the molecular mechanism of cooperativity in human hemoglobin cannot be limited to a first-order free energy coupling concept

Studies of the linkage between ligand binding and subunit assembly of oligomeric proteins have extensively used the concept of free energy coupling. The "order" of these free energy couplings was introduced [Weber, G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7098-7102] as the number of subunits that must be liganded to alter specific intersubunit interactions. This concept dictates that the ligation of fewer subunits has no effect, but once the order number of subunits becomes ligated, the specific intersubunit interaction energy between those particular subunits is completely eliminated. Weber's report claims that the free energy coupling between oxygen binding and the dimer-tetramer subunit assembly in stripped human hemoglobin A is "first order". This conclusion is based on the analysis of a set of previously published equilibrium constants [Mills, F. C., Johnson, M. L., & Ackers, G. K. (1976) Biochemistry 15, 5350-5362]. I subsequently reported that the original experimental data, from which the equilibrium constants were derived, are consistent with both the first-order and "second-order" free energy coupling concepts [Johnson, M. L. (1986) Biochemistry 25, 791-797]. I also demonstrated that more precise recent experimental data [Chu, A. H., Turner, B. W., & Ackers, G. K. (1984) Biochemistry, 23, 604-617] are consistent with both the first-order and second-order free energy coupling concepts. A recent article [Weber, G. (1987) Biochemistry 26, 331-332] disagrees that the oxygen-binding data for human hemoglobin A are consistent with a second-order model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy.

Analysis of the histidine-containing protein (HPr) from Escherichia coli by two-dimensional homonuclear and heteronuclear nuclear magnetic resonance techniques has been performed, extending the work originally reported [Klevit, R. E., Drobny, G. D., & Waygood, E. B. (1986) Biochemistry 25, 7760-7769; Klevit, R. E., & Drobny, G. P. (1986) Biochemistry 25, 7770-7773; Klevit, R. E., & Waygood, E. B. (1986) Biochemistry 25, 7774-7781]. Two-dimensional homonuclear total coherence spectroscopy (TOCSY) allowed for more complete assignments of the side-chain spin systems than had been possible in the original studies. As well, two-dimensional 15N-1H heteronuclear spectroscopy was used to resolve a number of ambiguities present in the homonuclear spectra due to resonance redundancies. These analyses led to the correction of a number of resonance assignments that were made with the spectra that could be collected with the technology that existed 6 years ago. In addition, amide exchange rates and 3JNH coupling constants have been measured, extending the original analysis and yielding new structural information. All these data have been used to reexamine the folding topology of E. coli HPr. Structure calculations showed that the topology derived from the earlier NMR data, i.e., a four-stranded beta-sheet with three alpha-helices running along one side of the sheet, was essentially unchanged, although at the present level of analysis, a well-defined "helix B" could not be established with high confidence. In addition, the data reported here revealed the existence of two slowly-exchanging side-chain hydroxyl protons belonging to Ser31 and Thr59. Their behavior strongly suggests that these side chains are involved in hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs

A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe [Brown, E. R., Coker, G. T., III, & O'Malley, K. L. (1987) Biochemistry 26, 5208-5212]. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping. Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase [DiLella, A. G., Kwok, S. C. M., Ledley, F. D., Marvit, J., & Woo, S. L. C. (1986) Biochemistry 25, 743-749] genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence.     

Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant

The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding

In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes [Jaynes, E. N., Jr., Grant, P. G., Giangrande, G., Wieder, R., & Cooperman, B. S. (1978) Biochemistry 17, 561-569; Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274], that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits [Kerlavage, A. R., Weitzmann, C. J., & Cooperman, B. S. (1984) J. Chromatogr. 317, 201-212]. In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation. We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline. Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation.     

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)