Comparative FTIR analysis of the microenvironment of in cyanide-treated, high pH-treated and iron-depleted photosystem II membrane fragments

FTIR difference spectra due to light-induced formation were measured in control PS II membrane fragments and in samples where the magnetic interaction with the non-heme iron center in its high-spin Fe2+ state is eliminated by three different methods, i.e., extraction of the non-heme iron center, treatment with cyanide, and incubation at high pH (pH = 11). The results obtained reveal that (i) the most pronounced band at 1479 cm-1 reflecting the C../--O stretching mode of in the semiquinone anion radical remains invariant to "iron depletion" while it shifts by 4 and 2 cm-1 to lower frequencies upon cyanide and high pH treatments, respectively, (ii) peaks observed in the 2600-3000 cm-1 region which arise from Fermi resonance of harmonics and combinations of the imidazole ring modes with the hydrogen-bonding NH stretching vibrations are not affected upon iron depletion but are lost in cyanide and high pH-treated samples, and (iii) all three treatments give rise to some similar changes in the amide I bands of the protein backbones and in imidazole ring modes of the coupled histidine. These results show that the hydrogen-bonding interaction of is virtually unaffected upon non-heme iron depletion; in particular, the strong hydrogen bond between QA and a histidine side chain (most likely His 215 of the D2 subunit) is not changed. In marked contrast, drastic changes take place in the hydrogen bonding between QA and His upon CN- and high pH treatments. The straightforward interpretation is that the hydrogen bond is lost upon these treatments. Despite the striking difference in the effect of hydrogen-bonding interaction, all three treatments lead to similar structural pertubations on the protein conformational changes due to formation and ring vibrations of the coupled histidine side chain. On the basis of the data presented in the study, it is inferred that, concerning the hydrogen bond interaction, the microenvironment of is close to the native state when a suitable iron depletion is performed. Accordingly, the previously reported conclusion on the hydrogen-bonding pattern of in PS II [MacMillan, F., Lendzian, F., Renger, G., and Lubitz, W. (1995) Biochemistry 34, 8144-8156] studied by using iron-depleted preparations most likely reflects the situation in an intact PS II.     

Mid- to low-frequency Fourier transform infrared spectra of S-state cycle for photosynthetic water oxidation in Synechocystis sp. PCC 6803

Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.     

Changes of low-frequency vibrational modes induced by universal 15N- and 13C-isotope labeling in S2/S1 FTIR difference spectrum of oxygen-evolving complex

The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand.     

Analysis of flash-induced FTIR difference spectra of the S-state cycle in the photosynthetic water-oxidizing complex by uniform 15N and 13C isotope labeling

Protein bands in flash-induced Fourier transform infrared (FTIR) difference spectra of the S-state cycle of photosynthetic water oxidation were analyzed by uniform (15)N and (13)C isotopic labeling of photosystem II (PS II). The difference spectra upon first- to fourth-flash illumination were obtained with hydrated (for the 1800-1200 cm(-)(1) region) or deuterated (for the 3500-3100 cm(-)(1) region) films of unlabeled, (15)N-labeled, and (13)C-labeled PS II core complexes from Thermosynechococcus elongatus. Shifts of band frequencies upon (15)N and (13)C labeling provided the assignments of major peaks in the regions of 3450-3250 and 1700-1630 cm(-)(1) to the NH stretches and amide I modes of polypeptide backbones, respectively, and the assignments of some of the peaks in the 1600-1500 cm(-)(1) region to the amide II modes of backbones. Other prominent peaks in the latter region and most of the peaks in the 1450-1300 cm(-)(1) region exhibited large downshifts upon (13)C labeling but were unchanged by (15)N labeling, and hence assigned to the asymmetric and symmetric COO(-) stretching vibrations, respectively, of carboxylate groups in Glu, Asp, or the C-terminus. Peak positions corresponded well with each other among the first- to fourth-flash spectra, and most of the bands in the first- and/or second-flash spectra appeared with opposite signs of intensity in the third- and/or fourth-flash spectra. This observation indicates that the protein movements in the S(1)-->S(2) and/or S(2)-->S(3) transitions are mostly reversed in the S(3)-->S(0) and/or S(0)-->S(1) transitions, representing a catalytic role of the protein moieties of the water-oxidizing complex. Drastic structural changes in carboxylate groups over the S-state cycle suggest that the Asp and/or Glu side chains play important roles in the reaction mechanism of photosynthetic water oxidation.     

FTIR detection of structural changes in a histidine ligand during S-state cycling of photosynthetic oxygen-evolving complex

Changes in structural coupling between the Mn cluster and a putative histidine ligand during the S-state cycling of the oxygen-evolving complex (OEC) have been detected directly by Fourier transform infrared (FTIR) spectroscopy in photosystem (PS) II core particles from the cyanobacterium Synechocystis sp. PCC6803, in which histidine residues were selectively labeled with l-[(15)N(3)]histidine. The bands sensitive to the histidine-specific isotope labeling appeared at 1120-1090 cm(-)(1) in the spectra induced upon the first-, second-, and fourth-flash illumination, for the S(2)/S(1), S(3)/S(2), and S(1)/S(0) differences, at similar frequencies with different sign and/or intensity depending on the respective S-state transitions. However, no distinctive band was observed in the third-flash induced spectrum for the S(0)/S(3) difference. The results indicate that a single histidine residue coupled with the structural changes of the OEC during the S-state cycling is responsible for the observed histidine bands, in which the histidine modes changed during the S(0)-to-S(1) transition are reversed upon the S(1)-to-S(2) and S(2)-to-S(3) transitions. The 1186(+)/1178(-) cm(-)(1) bands affected by l-[(15)N(3)]histidine labeling were observed only for the S(2)/S(1) difference, but those affected by universal (15)N labeling appeared prominently showing a clear S-state dependency. Possible origins of these bands and changes in the histidine modes during the S-state cycling are discussed.     

Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy

The redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc(1) complex was studied by analysis of the pH dependencies of redox difference spectra using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The ISP of Rhodobacter capsulatus within the intact cytochrome bc(1) complex was analyzed in a mutant form in which the midpoint potential of cytochrome c(1) was lower than that of the ISP. This was compared to a soluble domain of the ISP from the bovine bc(1) complex. Spectra of in situ bacterial and isolated bovine proteins differ markedly only in part of their amide I regions with the in situ protein having additional pH-dependent component(s). Apart from this, both in situ and isolated proteins exhibited the same pH-dependent IR features in reduced minus oxidized difference spectra. Specifically, at high pH, a strong H/D insensitive negative band appeared at 1447/1450 cm(-)(1), together with a peak at 1310 cm(-)(1), the change of a 1267/1255 cm(-)(1) peak/trough into a simple 1266 cm(-)(1) peak, and a trough at 1107 cm(-)(1). Comparison with spectra of model materials indicates that all four signals arise from an imidazolate to imidazole transition of histidine, hence providing the first direct demonstration that histidine is the redox-linked protonation site of the ISP. The 1450 cm(-)(1) band can be assigned to a ring stretch that is unique to the imidazolate form of histidine. It is relatively sharp, has a high extinction coefficient, and provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms generally.     

Redox control of proton transfers in membrane b-type cytochromes: an absorption and resonance Raman study on bis(imidazole) and bis(imidazolate) model complexes of iron-protoporphyrin

Optical absorption spectra and resonance Raman (RR) spectra, obtained with Soret excitation, are reported for bis(imidazole) and bis(imidazolate) complexes of iron(II)- and iron(III)-protoporphyrin IX, prepared in aqueous conditions. Perdeuteration experiments on the axial ligands permitted the assignment of the symmetric Fe-(ligand)₂ stretching mode of Fe[x]PP(L)₂ to RR bands at 203 (x = II; L = ImH), 212 (x = II; L = Im^–), 201 (x = III; L = ImH) and 226 cm^–1 (x = III; L = Im^–). These frequency differences indicate a strengthening of the axial bonds when the imidazole deprotonations occur. The larger difference observed for the ferric derivatives reflects the stronger -donor capability of the Im^– anion for iron(III) over iron(II). For the ferrous derivatives, the frequencies of several skeletal porphyrin modes (₄, ₁₀, ₁₁ and ₃₈) are downshifted by 2–10 cm^–1 upon deprotonation of the ligands. This effect corresponds to an increased back-bonding from the metal atom to the porphyrin ring when the axial ligand decreases its -acid strength. Bringing further support to this interpretation, an inverse linear relationship is established between the frequencies of (Fe(Il)-L₂) and ₁₁. This correlation is expected to monitor the overall H-bonding state of histidine ligands of reduced cytochromes b. On the other hand, absorption measurements have characterized large pK_a differences for the sequential imidazole ionizations of Fe[x]PP(ImH)₂ in aqueous cetyltrimethylammonium bromide (9.0 and 10.8 for x = 111; 13.0 and 14.1 for x = II). These titrations show that Fe(II)PP(Im^–)₂ and Fe(III)PP(ImH)₂ are good proton-acceptor and proton-donor, respectively, and suggest a model by which heme, located in a favorable environment inside a cytochrome, could couple a cycle of electron transfer with a proton transfer. Based on sequence data and structural models, it is further proposed that, in several membrane cytochromes b (b, b ₆, b ₅₅₉), a positively charged amino acid residue and an imidazolate ligand of the ferriheme could form an ion pair involved in a redox control of proton transfer.Abbreviations RR resonance Raman - EPR electron paramagnetic resonance - PP protoporphyrin IX - ImH imidazole - Im^– imidazolate - Im* imidazole or imidazolate - 1MeIm 1-methylimidazole - HisH histidine - His^– histidinate - CTABr cetyltrimethylammonium bromide - NaDS sodium dodecylsulphate - VLP very low potential - LP low potential - HP high potential     

Resonance Raman studies of Rieske-type proteins.

Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster.     

N-Isotope effects on the Raman spectra of Fe2S2 ferredoxin and Rieske ferredoxin: evidence for structural rigidity of metal sites

The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe(2)S(2) ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins. Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe(2)S(2) ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform (15)N-labeling of Anabaena Fe(2)S(2) ferredoxin results in shifts of -1 cm(-1) in the Fe-S(t) stretching modes at 282, 340, and 357 cm(-1). The (15)N dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen. No (15)N dependence occurs for the nu(Fe-S(b)) modes at 395 and 426 cm(-1). Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-monooxygenase system of Pseudomonas mendocina. Upon selective (15)N-labeling of the alpha-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm(-1) all undergo shifts of -1 to -2 cm(-1), thereby identifying them as combinations of nu(Fe-S(t)) and delta(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with (15)N-histidine ((15)N in either the alpha1,delta1 or delta1,epsilon2 positions). Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety. The extensive kinematic coupling of iron ligand vibrations observed in both the Fe(2)S(2) and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins. In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer.     

Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.     

Direct evidence that all three histidine residues coordinate to Cu(II) in amyloid-beta1-16

We provide direct evidence that all three histidine residues in amyloid-beta 1-16 (Abeta 1-16) coordinate to Cu(II). In our approach, we generate Abeta 1-16 analogues, in each of which a selected histidine residue is isotopically enriched with (15)N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopy clearly show that all three histidine imidazole rings at positions 6, 13 and 14 in Abeta 1-16 bind to Cu(II). The method employed here does not require either chemical side chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We find that the histidine coordination in the Abeta 1-16 peptide is independent of the Cu(II)-to-peptide ratio, which is in contrast to the Abeta 1-40 peptide. The ESR results also suggest tight binding between the histidine residues and the Cu(II) ion, which is likely the reason for the high binding affinity of the Abeta peptide for Cu(II).     

Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.     

Resonance Raman study on synergistic activation of soluble guanylate cyclase by imidazole, YC-1 and GTP

Soluble guanylate cyclase (sGC), a physiological nitric oxide (NO) receptor, is a heme-containing protein and catalyzes the conversion of GTP to cyclic GMP. We found that 200 mM imidazole moderately activated sGC in the coexistence with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), although imidazole or YC-1 alone had little effect for activation. GTP facilitated this process. Resonance Raman spectra of imidazole complex of native sGC and CO-bound sGC (CO-sGC) have demonstrated that a simple heme adduct with imidazole at the sixth coordination position is not present for both sGC and CO-sGC below 200 mM of the imidazole concentration and that the Fe-CO stretching band (nuFe-CO)) appears at 492 cm(-1) in the presence of imidazole compared with 473 cm(-1) in its absence. Both frequencies fall on the line of His-coordinated heme proteins in the nuFe-CO vs nuC-O plot. However, it is stressed that the CO-heme of sGC becomes apparently photo-inert in a spinning cell in the presence of imidazole, suggesting the formation of five-coordinate CO-heme or of six-coordinate heme with a very weak trans ligand. These observations suggest that imidazole alters not only the polarity of heme pocket but also the coordination structure at the fifth coordination side presumably by perturbing the heme-protein interactions at propionic side chains. Despite the fact that the isolated sGC stays in the reduced state and is not oxidized by O(2), sGC under the high concentration of imidazole (1.2 M) yielded nu4 at 1373 cm(-1) even after its removal by gel-filtration, but addition of dithionite gave the strong nu4 band at 1360 cm(-1). This indicated that imidazole caused autoxidation of sGC.     

Fourier transform infrared spectrum of the secondary quinone electron acceptor Q(B) in photosystem II

Fourier transform infrared difference spectra upon single reduction of the secondary quinone electron acceptor Q(B) in photosystem II (PSII), without a contribution from the electron donor-side signals, were obtained for the first time using Mn-depleted PSII core complexes of the thermophilic cyanobacterium Thermosynechococcus elongatus. The Q(B)(-)/Q(B) difference spectrum exhibited a strong C...O stretching band of the semiquinone anion at 1480 cm(-)(1), the frequency higher by 2 cm(-)(1) than that of the corresponding band of Q(A)(-), in agreement with the previous S(2)Q(B)(-)/S(1)Q(B) spectrum of the PSII membranes of spinach [Zhang, H., Fischer, G., and Wydrzynski, T. (1998) Biochemistry 37, 5511-5517]. Also, several peaks originating from the Fermi resonance of coupled His modes with its strongly H-bonded NH vibration were observed in the 2900-2600 cm(-)(1) region, where the peak frequencies were higher by 7-24 cm(-)(1) compared with those of the Q(A)(-)/Q(A) spectrum. These frequency differences suggest that H-bond interactions of the CO groups, especially with a His side chain, are different between Q(B)(-) and Q(A)(-). Furthermore, a prominent positive peak was observed at 1745 cm(-)(1) in the C=O stretching region of COOH or ester groups in the Q(B)(-)/Q(B) spectrum. The peak frequency was unaffected by D(2)O substitution, indicating that this peak does not arise from a COOH group but probably from the 10a-ester C=O group of the pheophytin molecule adjacent to Q(B). The absence of protonation of carboxylic amino acids upon Q(B)(-) formation in contrast to the previous observation in the purple bacterium Rhodobacter sphaeroides suggests that the protonation mechanism of Q(B) in PSII is different from that of bacterial reaction centers.     

Protonation and hydrogen-bonding state of the distal histidine in the CO complex of horseradish peroxidase as studied by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman (UVRR) spectroscopy has been used to characterize the structure and hydrogen bonding state of the distal histidine (His42) in horseradish peroxidase (HRP) complexed with carbon monoxide (HRP-CO). The HRP-CO - HRP UVRR difference spectrum in D(2)O solution at pD 7.0 shows two positive peaks at 1408 and 1388 cm(-)(1), which are ascribable to medium-to-weak and strong hydrogen bonding states, respectively, of the protonated imidazolium side chain of His42 in HRP-CO. Both His42 peaks decrease in intensity with increase of pD with a midpoint of transition at pD 8.8, indicating that the pK(a) of His42 in HRP-CO is 8.8. The CO ligand exhibits two C-O stretching Raman peaks at 1932 and 1902 cm(-)(1), the latter of which diminishes at alkaline pD and is assignable to a strong hydrogen-bonded state. It is most probable that the imidazolium side chain of His42 forms a strong hydrogen bond with CO, giving a His42 peak at 1388 cm(-)(1) and a CO peak at 1902 cm(-)(1), in one conformer. The other hydrogen bonding state of His42, giving the 1408 cm(-)(1) peak, is ascribed to another conformer forming a medium-to-weak hydrogen bond with a water molecule in the distal cavity. The present finding that His42 can act as a strong proton donor to CO and decrease the CO bond order is consistent with the role of His42 as a general acid to cleave the O-O bond of hydrogen peroxide, a specific oxidizing agent, in the catalytic cycle of HRP.     

Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.     

Heme pocket interactions in cytochrome c peroxidase studied by site-directed mutagenesis and resonance Raman spectroscopy

Resonance Raman spectra are reported for FeII and FeIII forms of cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis and cloning in Escherichia coli. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn, Trp-191----Phe) and distal (Trp-51----Phe, Arg-48----Leu and Lys) side of the heme. These spectra are used to assess the spin and ligation states of the heme, via the porphyrin marker band frequencies, especially v3, near 1500 cm-1, and, for the FeII forms, the status of the Fe-proximal histidine bond via its stretching frequency. The FeII-His frequency is elevated to approximately 240 cm-1 in CCP(MI) and in all of the distal mutants, due to hydrogen-bonding interactions between the proximal His-175 N delta and the carboxylate acceptor group on Asp-235. The FeII-His RR band has two components, at 233 and 246 cm-1, which are suggested to arise from populations having H-bonded and deprotonated imidazole; these can be viewed in terms of a double-well potential involving proton transfer coupled to protein conformation. The populations shift with changing pH, possibly reflecting structure changes associated with protonation of key histidine residues, and are influenced by the Leu-48 and Phe-191 mutations. A low-spin FeII form is seen at high pH for the Lys-48, Leu-48, Phe-191, and Phe-51 mutants; for the last three species, coordination of the distal His-52 is suggested by a approximately 200-cm-1 RR band assignable to Fe(imidazole)2 stretching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flash-induced Fourier transform infrared detection of the structural changes during the S-state cycle of the oxygen-evolving complex in photosystem II

Fourier transform infrared (FTIR) difference spectra of all flash-induced S-state transitions of the oxygen-evolving complex were measured using photosystem II (PSII) core complexes of Synechococcus elongatus. The PSII core sample was given eight successive flashes with 1 s intervals at 10 degrees C, and FTIR difference spectra upon individual flashes were measured. The obtained difference spectra upon the first to fourth flashes showed considerably different spectral features from each other, whereas the fifth, sixth, seventh, and eighth flash spectra were similar to the first, second, third, and fourth flash spectra, respectively. The intensities at the wavenumbers of prominent peaks of the first and second flash spectra showed clear period four oscillation patterns. These oscillation patterns were well fitted with the Kok model with 13% misses. These results indicate that the first, second, third, and fourth flash spectra represent the difference spectra upon the S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1) transitions, respectively. In these spectra, prominent bands were observed in the symmetric (1300-1450 cm(-)(1)) and asymmetric (1500-1600 cm(-)(1)) stretching regions of carboxylate groups and in the amide I region (1600-1700 cm(-)(1)). Comparison of the band features suggests that the drastic coordination changes of carboxylate groups and the protein conformational changes in the S(1) --> S(2) and S(2) --> S(3) transitions are reversed in the S(3) --> S(0) and S(0) --> S(1) transitions. The flash-induced FTIR measurements during the S-state cycle will be a promising method to investigate the detailed molecular mechanism of photosynthetic oxygen evolution.     

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.     

Carbon monoxide complex of cytochrome b(5) at acidic pH

CO complex of cyt b(5) generated at acidic pH is investigated by absorption, resonance Raman (RR), and far UV CD measurements. The Soret maximum wavelength blue-shifted to 420 nm with other absorption bands observed around 540 and 570 nm for reduced cyt b(5) upon interaction with CO at acidic pH (pH 3.1-3.5). Under this condition, the iron-carbon stretching RR band was observed at 529 cm(-1) (520 cm(-1) for C(18)O), which indicated formation of a heme&bond;CO adduct with a histidine as an axial ligand. Heme dissociated from the reduced cyt b(5) protein at pH approximately 3.5, whereas its rate decreased under CO atmosphere compared with N(2) atmosphere, due to formation of a heme&bond;CO adduct with a histidine as an axial ligand.