Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.     

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosines-26 and -64

Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR570----K630 or BR570----M412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K630 and the deprotonation of a tyrosine by M412 [Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M412 as concluded previously [Scherrer, P., & Stoeckenius, W. (1985) Biochemistry 24, 7733-7740]. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm-1 in the BR570----M412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue [Engelhard, M., Gerwert, K., Hess, B., Kreutz, W., & Siebert, F. (1985) Biochemistry 24, 400-407; Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.     

Site-directed mutagenesis in haemoglobin. Functional role of tyrosine-42(C7) alpha at the alpha 1-beta 2 interface

To clarify the functional role of Tyr-42(C7) alpha, which forms a hydrogen bond with Asp-99(G1) beta at the alpha 1-beta 2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42 alpha was replaced by Phe (Hb Phe-42 alpha) or His (Hb His-42 alpha), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99 beta mutants, Hb Phe-42 alpha showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42 alpha, the deoxy T quaternary structure is highly destabilized and the strain imposed on the Fe-N epsilon (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42 alpha, Hb His-42 alpha showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42 alpha and Asp-99 beta. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42 alpha and Asp-99 beta plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.     

Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle

The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.     

Ultraviolet resonance Raman and absorption difference spectroscopy of myoglobins: titration behavior of individual tyrosine residues

The UV resonance Raman spectra of horse and sperm whale myoglobin excited at 240 nm show bands between 600 and 1700 cm-1 which derive from tyrosyl and tryptophyl residues. No significant contribution from phenylalanine and peptide backbone vibrations occurs at this excitation wavelength. We examine the pH dependence of the UV resonance Raman and UV absorption difference spectra of these myoglobins to correlate the local protein environment of the tyrosyl residues as given by the protein crystal structure to their pKa values, molar absorptivities, and Raman cross sections. Some of our pKa values for the tyrosinate residues of horse Mb differ from those of previous studies. We show that the lambda max values, the molar absorptivities, and the Raman cross sections are sensitive to the local environment of the tyrosinate residues in the protein. We relate differences in the tyrosyl absorption spectra to differences in Raman cross sections. In addition, we discuss the importance to the Raman cross sections of the local electromagnetic field enhancement due to the dielectric environment of the tyrosinate residues in the protein. This local field should scale the Raman cross sections in a way useful as a probe of the average aromatic amino acid residue environment.     

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.     

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.     

Molecular dynamics study of the M412 intermediate of bacteriorhodopsin.

Molecular dynamics simulations have been carried out to study the M412 intermediate of bacteriorhodopsin's (bR) photocycle. The simulations start from two simulated structures for the L550 intermediate of the photocycle, one involving a 13-cis retinal with strong torsions, the other a 13,14-dicis retinal, from which the M412 intermediate is initiated through proton transfer to Asp-85. The simulations are based on a refined structure of bR568 obtained through all-atom molecular dynamics simulations and placement of 16 waters inside the protein. The structures of the L550 intermediates were obtained through simulated photoisomerization and subsequent molecular dynamics, and simulated annealing. Our simulations reveal that the M412 intermediate actually comprises a series of conformations involving 1) a motion of retinal; 2) protein conformational changes; and 3) diffusion and reconfiguration of water in the space between the retinal Schiff base nitrogen and the Asp-96 side group. (1) turns the retinal Schiff base nitrogen from an early orientation toward Asp-85 to a late orientation toward Asp-96; (2) disconnects the hydrogen bond network between retinal and Asp-85 and tilts the helix F of bR, enlarging bR's cytoplasmic channel; (3) adds two water molecules to the three water molecules existing in the cytoplasmic channel at the bR568 stage and forms a proton conduction pathway. The conformational change (2) of the protein involves a 60 degrees bent of the cytoplasmic side of helix F and is induced through a break of a hydrogen bond between Tyr-185 and a water-side group complex in the counterion region.     

Picosecond structural dynamics of myoglobin following photodissociation of carbon monoxide as revealed by ultraviolet time-resolved resonance Raman spectroscopy

Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds.     

Inter- and intramolecular disulfide bond formation and related structural changes in the lens proteins. A Raman spectroscopic study in vivo of lens aging

Raman spectra have been measured for intact rat lens nuclei at various stages of aging in an attempt to gain further insight into age-related structural changes in the lens proteins, especially changes concerning protein sulfhydryl groups. Two Raman bands at 2579 and 2561 cm-1 were observed to be assignable to SH stretching modes of the cysteine residues. These bands have been attributed to "exposed" and "buried" sulfhydryl groups of the lens proteins, respectively, on the basis of a model compound study. The relative intensities of both SH stretching modes decreased with lens aging, and concurrently the intensity of a S-S stretching mode at 509 cm-1 due to disulfide bridges increased, suggesting that not only exposed but also buried protein sulfhydryl groups are converted to disulfide groups as a result of aging. The rate of the intensity decrease in the 2561 cm-1 band was similar to that in the 2579 cm-1 band. Therefore, it seems likely that the sulfhydryl groups in the two distinct environments are nearly equally subjected to the oxidation. Cysteine and cystine residues of the lens proteins gave their C-S stretching modes at 708 cm-1, indicating that they predominantly assume PC and/or PN conformers. The intensity ratio of a tyrosine doublet near 840 cm-1 (I832/I855) changed from approximately 0.86 to approximately 0.81 with the aging of the rat lens. This result implies that some tyrosine residues undergo a change in their hydrogen bonding environments during the course of aging. Of particular importance is that the relative intensity change of the tyrosine doublet with normal aging and that with cataract formation are in opposite directions.     

Mechanism of light-dependent proton translocation by bacteriorhodopsin.

Site-specific mutagenesis has identified amino acids involved in bR proton transport. Biophysical studies of the mutants have elucidated the roles of two membrane-embedded residues: Asp-85 serves as the acceptor for the proton from the isomerized retinylidene Schiff base, and Asp-96 participates in reprotonation of this group. The functions of Arg-82, Leu-93, Asp-212, Tyr-185, and other residues that affect bR properties when substituted are not as well understood. Structural characterization of the mutant proteins will clarify the effects of substitutions at these positions. Current efforts in the field remain directed at understanding how retinal isomerization is coupled to proton transport. In particular, there has been more emphasis on determining the structures of bR and its photointermediates. Since well-ordered crystals of bR have not been obtained, continued electron diffraction studies of purple membrane offer the best opportunity for structure refinement. Other informative techniques include solid-state nuclear magnetic resonance of isotopically labeled bR (56) and electron paramagnetic resonance of bR tagged with nitroxide spin labels (2, 3, 13, 15). Site-directed mutagenesis will be essential in these studies to introduce specific sites for derivatization with structural probes and to slow the decay of intermediates. Thus, combining molecular biology and biophysics will continue to provide solutions to fundamental problems in bR.     

Insights into how CUB domains can exert specific functions while sharing a common fold: conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.     

Effects of amino acid substitutions in the F helix of bacteriorhodopsin. Low temperature ultraviolet/visible difference spectroscopy

Site-specific mutagenesis in combination with low temperature UV/visible difference spectroscopy has been used to investigate the role of individual amino acids in the structure and function of bacteriorhodopsin (bR). We examined the effects of eight single amino acid substitutions, all in the putative F helix, on the absorption of bR as well as formation of the K and M intermediates. Both the absorbance spectra and the photocycle difference spectra of Escherichia coli expressed bR as well as the mutants S183A, P186G, and E194Q all closely resembled the corresponding purple membrane spectra. In contrast the Pro-186----Leu substitution resulted in the loss of the normal photocycle and a large blue shift in the bR state lambda max. Thus, Pro-186 appears to play a critical role in maintaining the normal protein-chromophore interactions, although the pyrrolidine ring is not essential since proline could be replaced by glycine at this position. The mutants W182F, W189F, and S193A did not appear to be directly involved in the bathochromic shift of bR since they all had lambda max's close to that of purple membrane and produced intermediates similar to K and M. However, alterations in the UV and visible difference spectra as well as the appearance of some irreversibility in the photoreactions indicate that these mutants have altered protein-chromophore interactions during the photocycle. Unlike the other mutants examined, Y185F exhibited a red-shifted form of bR and K raising the possibility that Tyr-185 is directly involved in color regulation. In addition, UV difference peaks previously associated with a tyrosine deprotonation were absent in Y185F indicating that Tyr-185 undergoes protonation changes during the photocycle in agreement with recent Fourier transform infrared difference measurements (Braiman, M.S., Mogi, T., Stern, L. J., Hackett, N., Chao, B. H., Khorana, H.G., and Rothschild, K. J. (1988) Proteins: Structure, Function, and Genetics 3, 219-229). Our results suggest that Trp-182, Tyr-185, Pro-186, Trp-189, and Ser-193, all of which are within a 100 degrees segment of the F helix, are part of a retinal-binding pocket.     

Bacteriorhodopsin's M412 intermediate contains a 13-cis, 14-s-trans, 15-anti-retinal Schiff base chromophore

The structure of the retinal chromophore about the C = N and C14-C15 bonds in bacteriorhodopsin's M412 intermediate has been determined by analyzing resonance Raman spectra of 2H and 13C isotopic derivatives. Normal mode calculations on 13-cis-retinal Schiff bases demonstrate that the C15-D rock and N-CLys stretch are strongly coupled for C = N-syn chromophores and weakly coupled for C = N-anti chromophores. When the Schiff base geometry is anti, the C15-D rock appears as a localized resonance Raman active mode at approximately 980 cm-1, which is moderately sensitive to 13C substitution at positions 14 and 15 (approximately 7 cm-1) and insensitive to 13C substitution at the epsilon position of lysine. When the Schiff base geometry is syn, in-phase and out-of-phase combinations of the C15-D rock and N-CLys stretch are predicted at approximately 1060 and approximately 910 cm-1, respectively. The in-phase mode is more sensitive to 13C substitution at positions 14 and 15 (approximately 15 cm-1) and at the epsilon position of lysine (approximately 4 cm-1). Calculations and comparison with experimental data on dark-adapted bacteriorhodopsin indicate that the in-phase mode at approximately 1060 cm-1 carries the majority of the resonance Raman intensity. M412 exhibits a C15-D rock at 968 cm-1 that shifts 8 cm-1 when 13C is added at positions 14 and 15 and is insensitive to 13C substitution at the epsilon-position of lysine. This demonstrates that M412 contains a C = N-anti Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase. A sequence comparison

The small subunit of ribonucleoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center. An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula solidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important. Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved. We propose that Tyr-122 is responsible for radical stabilization and that His-118 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center.     

Comparative spectral analysis of mammalian, fungal, and bacterial catalases. Resonance Raman evidence for iron-tyrosinate coordination

Resonance Raman spectra are reported for catalases from bovine liver, the ascomycete fungus Aspergillus niger, and the bacterium Micrococcus luteus. The vibrational frequencies of the oxidation-, spin-, and coordination number-sensitive spectral bands are indicative of high spin pentacoordinate hemes in the resting ferric enzymes of each of these organisms. This result is in accord with the crystal structure of bovine catalase (Fita, I., and Rossmann, M.G. (1985) J. Mol. Biol. 185, 21-37). In contrast, the crystallographic study of catalase from the ascomycete Penicillium vitale (Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A.A., Grebenko, A. I., Borisov, V. V., Bartels, K. S., Fita, I., and Rossmann, M. G. (1986) J. Mol. Biol. 188, 49-61) showed electron density on the distal side of the heme which could imply the presence of a sixth ligand, possibly a water molecule. However, both of these crystallographic studies showed the proximal ligand in catalase to be a tyrosine. The present study confirms tyrosinate coordination in each of the three catalases from the appearance of selected resonance-enhanced tyrosine vibrational modes. The most characteristic band is the tyrosinate ring mode at approximately 1612 cm-1 which is maximally enhanced with 488.0 nm excitation. The appearance of tyrosinate modes at 1607 and 1245 cm-1 in the resonance Raman spectra of M. luteus cyano catalase serves to identify tyrosine as an axial ligand in bacterial as well as eukaryotic catalases. Unlike non-heme iron tyrosinate proteins, whose resonance Raman spectra are dominated by several intense bands diagnostic of tyrosine ligation, the heme-linked tyrosine modes are not easily distinguished from the large number of porphyrin vibrations.     

Interpretation of the doublet at 850 and 830 cm-1 in the Raman spectra of tyrosyl residues in proteins and certain model compounds.

The doublet at 850 and 830 cm-1 in the Raman spectra of proteins containing tyrosyl residues has been examined as to its origin and the relation of its components to the environment of the phenyl ring, the state of the phenolic hydroxyl group, and the conformation of the amino acid backbone. Raman spectral studies on numerous model molecules related to tyrosine, including certain deuterium derivatives, show that the doublet is due to Fermi resonance between the ring-breathing vibration and the overtone of an out-of-plane ring-bending vibration of the para-substituted benzenes. Further examination of the effects of pH and solvents on the Fermi doublet and of the crystallographic data demonstrates that the intensity ratio of the two components depends on changes in the relative frequencies of the two vibrations. These in turn are found to be sensitive to the nature of the hydrogen bonding of the phenolic hydroxyl group of its ionization, but much less so to the environment of the phenyl ring and the conformation of the amino acid backbone. By use of the relative intensities of the doublet in model systems where the phenolic hydroxyl group is strongly hydrogen-bonded, weakly hydrogen-bonded, free or ionized, the reported Raman intensities of the doublets observed in the Raman spectra of several proteins have been interpreted. The results are compared with those obtained by other techniques.     

Two processes lead to a stable all-trans and 13-cis isomer equilibrium in dark-adapted bacteriorhodopsin; effect of high pressure on bacteriorhodopsin,bacteriorhodopsin mutant D96N and fluoro-bacteriorhodopsin analogues

The combination of absorption spectroscopy and extraction techniques was applied to study the effect of high pressure on the dark-adapted state of bacteriorhodopsin, 14-(12-,10-)fluoro-bacteriorhodopsin, a D96N bacteriorhodopsin mutant, and 14-(12-,10-)fluoro-D96N. Evidence is presented that, at high pressure, the isomers' equilibrium is shifted from all- trans isomers towards the 13-cis isomers. Two groups of values for calculated molar volume changes indicate that there are at least two different processes leading to a stable all-trans and 13-cis isomers' equilibrium called the dark-adapted bacteriorhodopsin. The first process may be attributed to changes in the distances and rearrangement of functionally important residues and a retinal Schiff base. It is suggested that the moved residues (probably Asp-212 with the contribution of Tyr-185 and/or Asp-85) closer to the chromophore could catalyse its trans-cis isomerization. These changes require smaller pressure changes and induce larger volume changes (large-volume-change process). The second process may be attributed to the formation of the three hydrogen bonds that additionally decrease the volume and strengthen further stabilization of the 13-cis isomer. To induce these changes, larger changes of pressure are required and the final molar volume changes are smaller (small-volume-change process). The total molar volume change between all-trans bacteriorhodopsin and 13-cis bacteriorhodopsin in the dark-adapted state of native bacteriorhodopsin was found to be about -28 mL/mol, which is much higher than the value of about -7 mL/mol obtained previously (Tsuda and Ebrey 1980, Schulte and Bradley 1995). The data provide a novel insight into factors leading to stable isomer equilibrium in dark-adapted bacteriorhodopsin.