Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis

Sensory rhodopsin II is a seven transmembrane helical retinal protein and functions as a photoreceptor protein in negative phototaxis of halophilic archaea. Sensory rhodopsin II from Natronomonas pharaonis (NpSRII) is stable under various conditions and can be expressed functionally in Escherichia coli cell membranes. Rhodopsins from microorganisms, known as microbial rhodopsins, exhibit a photocycle, and light irradiation of these molecules leads to a high-energy intermediate, which relaxes thermally to the original pigment after passing through several intermediates. For bacteriorhodopsin (BR), a light-driven proton pump, the photocycle is established as BR → K → L → M → N → O → BR. The photocycle of NpSRII is similar to that of BR except for N, i.e., M thermally decays into the O, and N has not been well characterized in the photocycle. Thus we here examined the second half of the photocycle in NpSRII, and in the present transient absorption study we found the formation of a new photointermediate whose absorption maximum is ～500 nm. This intermediate becomes pronounced in the presence of azide, which accelerates the decay of M. Transient resonance Raman spectroscopy was further applied to demonstrate that this intermediate contains a 13-cis retinal protonated Schiff base. However, detailed analysis of the transient absorption data indicated that M-decay does not directly produce N but rather produces O that is in equilibrium with N. These observations allowed us to propose a structural model for a photocycle that involves N.     

Time-resolved methods in Biophysics. 1. A novel pump and probe surface-enhanced resonance Raman approach for studying biological photoreceptors.

This article describes a method, based on surface-enhanced resonance Raman (SERR) spectroscopy, for studying the reaction dynamics of photoreceptors immobilized on metal electrodes. Time resolution and fresh sample conditions are achieved by synchronizing the rotational and translational motion of a novel kinematic electrode with the duration and time delay between the pump and probe events. The power and sensitivity of the method is illustrated by studying the photocycle of the sensory photoreceptor NpSRII and its sensitivity to the applied electric field. The results are compared with time-resolved resonance Raman measurements in solution.     

Effects of solubilization on the structure and function of the sensory rhodopsin II/transducer complex

Lipid-protein interactions are known to play a crucial role in structure and physiological activity of integral membrane proteins. However, current technology for membrane protein purification necessitates extraction from the membrane into detergent micelles. Also, due to experimental protocols, most of the data available for membrane proteins is obtained using detergent-solubilized samples. Stable solubilization of membrane proteins is therefore an important issue in biotechnology as well as in biochemistry and structural biology. An understanding of solubilization effects on structural and functional properties of specific proteins is of utmost relevance for the evaluation and interpretation of experimental results. In this study, a comparison of structural and kinetic data obtained for the archaebacterial photoreceptor/transducer complex from Natronomonas pharaonis (NpSRII/NpHtrII) in detergent-solubilized and lipid-reconstituted states is presented. Laser flash photolysis, fluorescence spectroscopy, and electron paramagnetic resonance spectroscopy data reveal considerable influence of solubilization on the photocycle kinetics of the receptor protein and on the structure of the transducer protein. Especially the protein-membrane proximal region and the protein-protein interfacial domains are sensitive towards non-native conditions. These data demonstrate that relevance of biochemical and structural information obtained from solubilized membrane proteins or membrane protein complexes has to be evaluated carefully.     

Resonance Raman evidence for two conformations involved in the L intermediate of photoactive yellow protein

The blue light receptor photoactive yellow protein (PYP) displays a photocycle that involves several intermediate states. Here we report resonance Raman spectroscopic investigations of the short-lived red-shifted intermediate denoted PYP(L). We have found that the Raman bands of the carbonyl C=O stretching mode nu(11) as well as the C=C stretching mode nu(13) for the chromophore can be resolved into two peaks, and the ratio of the two components varies as a function of pH with pK(a) approximately 6. The isotope effects on the resonance Raman spectra have confirmed a deprotonated cis-chromophore for the two components. The results indicate the presence of two conformations in the active site of PYP(L). The normal coordinate calculations based on the density functional theory provide a structural model for the two conformations, where the low pH form is possibly an active structure for the protonation reaction generating a following intermediate in the photocycle.     

Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 +/- 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 +/- 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13 = C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode to 1618 cm-1 in D2O indicates that the Schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C = N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase

When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping.     

Resonance Raman spectroscopy of sensory rhodopsin II from Natronobacterium pharaonis

Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII(500) whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII(500) and the long-lived intermediates M(400), N(485) and O(535) based on the correlation between the C = C stretching frequency and the absorption maximum. The parent state and the intermediates N(485) and O(535) exhibit a protonated Schiff base. The C = N stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII(500) and O(535) whereas these interactions are most likely very weak in N(485).     

Dynamics of Rhodobacter capsulatus [2FE-2S] ferredoxin VI and Aquifex aeolicus ferredoxin 5 via nuclear resonance vibrational spectroscopy (NRVS) and resonance Raman spectroscopy

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(2)S(2)(Cys)(4) sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe-S stretching modes peaking around 335 cm(-1), and additional features observed as high as the B(2u) mode at approximately 421 cm(-1). Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B(3u) mode at approximately 288 cm(-1), attributed to an asymmetric combination of the two FeS(4) breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS(4) breathing. NRVS and RR analysis of isotope shifts with (36)S-substituted into bridging S(2-) ions in Rc FdVI allowed quantitation of S(2-) motion in different normal modes. We observed the symmetric Fe-Fe stretching mode at approximately 190 cm(-1) in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields. Progressively more complex D(2h) Fe(2)S(2)S'(4), C(2h) Fe(2)S(2)(SCC)(4), and C(1) Fe(2)S(2)(Cys)(4) models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe-S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated for analysis of data on the reduced FdVI. Finally, the degree of collectivity was used to quantitate the delocalization of the dynamic properties of the redox-active Fe site. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.     

Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mutations at tyrosine M210

Resonance Raman (RR) spectroscopy and low-temperature absorption spectroscopy have been used to investigate the structural changes in the reaction centers (RCs) of Rhodobacter sphaeroides induced by site-specific mutations on the tyrosine (Y) M210 residue. RCs in which Y M210 has been genetically replaced with phenylalanine (F) or leucine (L) exhibit a 5-fold decrease in their primary electron-transfer kinetics (Finkele et al., 1990). The general similarity of RR spectra of the wild-type RCs as compared to those of the two mutant RCs indicates that no significant global structural changes occur upon mutation at the level of any of the six bacteriochlorin pigments. In the RR spectra of the two mutant RCs there is a conspicuous absence of contributions from the BPheM prosthetic group, which is interpreted in terms of a change in the resonance enhancement conditions of this chromophore. Low-temperature adsorption spectroscopy reveals marked shifts in the Qx absorption band of BPheM. This shift is interpreted as arising from a destabilization of the protein in the vicinity of BPheM and accounts for the change in resonance condition for this chromophore in its RR contributions. As well, there is a 3-nm red shift of the Qy absorption band of the BChls from 803 to 806 nm for the mutant RCs. Difference RR spectra yielding structural information concerning, selectively, the primary donor (P) indicate that the structure of the P binding pocket is conserved for these mutant RCs. The tyrosine M210 is not observed to be engaged in a hydrogen bond with either of the acetyl or keto carbonyls of P.     

Resonance Raman spectroscopy and quantum chemical calculations reveal structural changes in the active site of photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.     

Procatalytic ligand strain. Ionization and perturbation of 8-nitroxanthine at the urate oxidase active site

The binding of the inhibitor 8-nitroxanthine to urate oxidase has been investigated by Raman and UV-visible absorption spectroscopy. The absorption maximum of 8-nitroxanthine shifts from 380 to 400 nm upon binding to the enzyme, demonstrating that the electronic structure of the ligand is perturbed. It has been proposed that oxidation of the substrate urate by urate oxidase is facilitated by formation of the substrate dianion at the enzyme active site, and Raman spectra of urate oxidase-bound 8-nitroxanthine suggest that both the dianionic and monoanionic forms of the ligand are bound to the enzyme under conditions where in solution the monoanion is present exclusively. The C4-C5 stretching frequency appears as a relatively isolated vibrational mode in 8-nitroxanthine whose frequency shifts according to the protonation state of the purine ring. Identification of the C4-C5 stretching mode was confirmed using [4-(13)C]-8-nitroxanthine and ab initio calculation of the vibrational modes. Two peaks corresponding to the C4-C5 stretching mode were evident in spectra of enzyme-bound 8-nitroxanthine, at 1541 and 1486 cm(-)(1). The higher frequency peak was assigned to monoanionic 8-nitroxanthine, and the low-frequency peak was assigned to dianionic 8-nitroxanthine. The C4-C5 stretching frequency for free monoanionic 8-nitroxanthine was at 1545 cm(-)(1), indicating that the enzyme polarizes that bond when the ligand is bound. The C4-C5 stretching frequency in dianionic 8-nitroxanthine is also shifted by 4 cm(-)(1) to lower frequency upon binding. For 8-nitroxanthine free in solution, the C4-C5 stretching frequency shifts to lower frequency upon deprotonation, and the absorption maximum in the UV-visible spectrum shifts to higher wavelength. The spectral shifts observed upon binding of 8-nitroxanthine to urate oxidase are consistent with increased anionic character of the ligand, which is expected to promote catalysis in the reaction with the natural substrate urate. In the Raman spectra of 8-nitroxanthine bound to the F179A, F179Y, and K9M mutant proteins, the C4-C5 stretching frequency was not perturbed from its position for the unbound ligand. Both V(max) and V/K were decreased in the mutant enzymes, demonstrating a correlation between the interaction that perturbs the C4-C5 stretching frequency and the catalytic activity of the enzyme. It is suggested that hydrogen-bonding interactions that lead to precise positioning and deprotonation of the substrate are perturbed by the mutations.     

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.     

Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps

Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (nu(Fe)(-)(CO)) of the CO complex and Fe(3+)-S stretching mode (nu(Fe)(-)(S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The nu(Fe)(-)(CO) mode was upshifted from 477 to 485 and to 490 cm(-)(1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas nu(Fe)(-)(CO) was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of nu(Fe)(-)(S) by 5 cm(-)(1) compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe(4+)=O porphyrin(+)(*)) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.     

Dynamics of the [4Fe-4S] cluster in Pyrococcus furiosus D14C ferredoxin via nuclear resonance vibrational and resonance Raman spectroscopies, force field simulations, and density functional theory calculations

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ～400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.     

A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

Resonance Raman investigation of the interaction of thromboxane synthase with substrate analogues

Thromboxane synthase is a hemethiolate enzyme that catalyzes the isomerization of prostaglandin H2 to thromboxane A2. We report the first resonance Raman (RR) spectra of recombinant human thromboxane synthase (TXAS) in both the presence and the absence of substrate analogues U44069 and U46619. The resting enzyme and its U44069 complex are found to have a 6-coordinate, low spin (6c/ls) heme, in agreement with earlier experiments. The U46619-bound enzyme is detected as a 6c/ls heme too, which is in contradiction with a previous conclusion based on absorption difference spectroscopy. Two new vibrations at 368 and 424 cm(-1) are observed upon binding of the substrate analogues in the heme pocket and are assigned to the second propionate and vinyl bending modes, respectively. We interpret the changes in these vibrational modes as the disruption of the protein environment and the hydrogen-bonding network of one of the propionate groups when the substrate analogues enter the heme pocket. We use carbocyclic thromboxane A2 (CTA2) to convert the TXAS heme cofactor to its 5-coordinate, high spin (5c/hs) form, as is confirmed by optical and RR spectroscopy. In this 5c/hs state of the enzyme, the Fe-S stretching frequency is determined at 350 cm(-1) with excitation at 356.4 nm. This assignment is supported by comparison to the spectrum of resting enzyme excited at 356.4 nm and by exciting at different wavelengths. Implications of our findings for substrate binding and the catalytic mechanism of TXAS will be discussed.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

Resonance Raman evidence for the presence of two heme pocket conformations with varied activities in CO-bound bovine soluble guanylate cyclase and their conversion

Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been classified as two types: sGC(1) and sGC(2). Here we demonstrate that the RR spectra of sGC isolated from bovine lung contain only sGC(2) while both species are observed in the spectra of the CO-bound form (CO-sGC). The relative populations of the two forms were altered from an initial composition in which the CO-sGC(2) form predominated, with the Fe-CO (nu(Fe)(-)(CO)) and C-O stretching modes (nu(CO)) at 472 and 1985 cm(-)(1), respectively, to a composition dominated by the CO-sGC(1) form with nu(Fe)(-)(CO) and nu(CO) at 488 and 1969 cm(-)(1), respectively, following the addition of a xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a significantly weaker effect, while a substrate analogue, GTP-gamma-S, had an effect similar to that of GTP. In contrast, ATP had a reverse effect, and suppressed the effects of YC-1 and GTP. In the presence of both YC-1 and GTP, vinyl vibrations of heme were significantly influenced. New CO isotope-sensitive bands were observed at 521, 488, 363, and 227 cm(-)(1). The 521 cm(-)(1) band was assigned to the five-coordinate (5c) species from the model compound studies using ferrous iron protoporphyrin IX in CTAB micelles. Distinct from the 472 cm(-)(1) species, both the 488 and 521 cm(-)(1) species were apparently un-photodissociable when an ordinary Raman spinning cell was used, indicating rapid recombination of photodissociated CO. On the basis of these findings, binding of YC-1 to the heme pocket is proposed.     

Resonance Raman applications in investigations of cytochrome c oxidase

Recent applications of resonance Raman (RR) spectroscopy in investigations of cytochrome c oxidase (CcO) are reviewed. Red-excited RR spectra for the fully oxidized "as-isolated" CcO tuned to the ligand-to-metal charge transfer absorption band at 655nm exhibit a Raman band at 755cm(-1) assignable to the ν(OO) stretching mode of a peroxide. Binding of CN(-) diminishes the RR band concomitant with the loss of the charge transfer absorption band. This suggests that a peroxide forms a bridge between heme a(3) and Cu(B). Time-resolved RR spectroscopy of whole mitochondria identified a band at 571cm(-1) arising from the oxygenated intermediate at Δt=0.4, 0.6 and 1.4ms. Bands at 804 and 780cm(-1) of the P and F intermediates were observed at Δt=0.6 and 1.4ms, respectively. The coordination geometries of the three intermediates are essentially the same as the respective species observed for solubilized CcO. However, the lifetime of the oxygenated intermediate in mitochondria was significantly longer than the lifetime of this intermediate determined for solubilized CcO. This phenomenon is due either to the pH effect of mitochondrial matrix, the effect of ΔpH and/or ΔΨ across the membrane, or the effect of interactions with other membrane components and/or phospholipids.