No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Axial ligands of chloroplast cytochrome b-559: Identification and requirement for a heme-cross-linked polypeptide structure
Abstract
Optical, resonance Raman, and electron paramagnetic resonance spectroscopies have been used to characterize the ligands and spin state of the chloroplast cytochrome b-559. The protein was isolated from both maize and spinach in a low-potential form. The spectroscopic data indicate that the heme iron in both ferric and ferrous cytochrome b-559 is in its low-spin state and ligated in its fifth and sixth coordination positions by histidine nitrogens. Electron paramagnetic resonance data for the purified spinach cytochrome are in good agreement with those determined by Bergström and Vänngård [Bergström, J., & Vänngård, T. (1982) Biochim. Biophys. Acta 682, 452-456] for a low-potential membrane-bound form of cytochrome b-559. The g values of high-potential cytochrome b-559 are shifted from those of its low-potential forms; this shift is interpreted as arising from a deviation of the planes of the two axial histidine imidazole rings from a parallel orientation. The model is consistent with the physical data and may also account for the facility with which cytochrome b-559 can be converted between low- and high-potential forms. Recent biochemical and molecular biological data [Widger, W. R., Cramer, W. A., Hermodson, M., Meyer, D., & Gullifor, M. (1984) J. Biol. Chem. 259, 3870-3876; Herrmann, R. G., Alt, J., Schiller, D., Cramer, W. A., & Widger, W. R. (1984) FEBS Lett. 179, 239-244] have shown that two polypeptides, one with 83 residues and a second with 39 residues, most likely constitute the protein of the cytochrome.(ABSTRACT TRUNCATED AT 250 WORDS)
... The only potential heme ligands in this region are histidine residues, namely His E23 in the '-protein and His F24 in the "-protein (highlighted in orange in Fig. 8.2). Indeed, it was shown by combining optical, resonance Raman (RR), and electron paramagnetic resonance (EPR) spectroscopy, that the fifth and sixth ligands to the heme iron of cyt b 559 are histidine nitrogens (Babcock et al. 1985) indicating that the whole heme protein consists of at least two '-helical subunits. The importance of a proper heme ligation was demonstrated by site-directed mutagenesis: The replacement of either E23 or F24 by Leu resulted in complete loss of PSII activity in S. 6803 (Pakrasi et al. 1991). ...
... The two subunits could be distinguished, because the C-terminus of PsbE extends significantly into the lumen as known from the amino acid sequence (Fig. 8.2). The electron density was consistent with an orientation of theplane of the heme group perpendicular to the membrane plane in agreement with EPR data (Bergström and Vänngard 1982;Crowder et al. 1982) and models based on the assignment of the axially ligating histidines (Babcock et al. 1985). What was new, however, was the observation, that the two TMHs of PsbE and PsbF are tilted with respect to the membrane normal and with respect to each other, giving the impression that they form a pair of tongs holding the heme group (see Figs. 8.3a and 8.4). ...
... There is now doubt that the axial ligands of the central iron ion influence the redox potential of the heme group. According to earlier spectroscopic work (Babcock et al. 1985) and the crystal structures, cyt b 559 has a typical bis-histidine ligation. The question is, whether this ligation is changed in a transition from one to another redox potential form of cyt b 559 . ...
The photosystem II core complex (PSIIcc) is a complicated membrane protein occurring in oxygenic photosynthetic organisms that catalyzes a key reaction, the light-induced oxidation of water. It consists of 17 membrane-spanning α-helical and three membrane-extrinsic protein subunits and binds nearly 100 cofactors. Among them is a heme protein composed of one heme b group ligated by two low molecular weight subunits with one transmembrane helix each, referred to as cytochrome (cyt) b
559. Despite extensive research, the role of cyt b
559 in PSIIcc is still not known. In the present chapter, the structure of cyt b
559 is reviewed based on recent crystallographic work together with its unusually complex redox behavior, and some ideas are discussed concerning a possible function of this unique heme protein.
... Cytb 559 is a heme-bridged heterodimer protein that is comprised of 1 α and 1 β subunit (subunits PsbE and PsbF encoded by psbE and psbF, respectively) (Umena et al., 2011;review by Müh and Zouni, 2015). Each subunit provides a histidine ligand for the non-covalently bound heme, which is located near the cytoplasmic side of PSII (Babcock et al., 1985). In contrast, most mono-heme cytochromes are made of a single polypeptide (Majumder and Blankenship, 2015). ...
... Structural determinants of the different redox-potential forms of Cytb 559 are still not clear. Previous studies suggested that the different redox-potential forms may be due to changes in hydrophobicity of the heme ligation environment (Krishtalik et al., 1993;Roncel et al., 2003), mutual orientation of the planes of histidine heme ligands (Babcock et al., 1985), or protonation or H-bonding pattern of the heme ligation environment (Ortega et al., 1988;Berthomieu et al., 1992;Roncel et al., 2001). A recent cryo-electron microscopy (cryo-EM) study presented a 1.95-Å resolution structural model of the native PSII preparation (PSII-D) from Thermosynechococcus, expected to predominantly feature the HP form of Cytb 559 . ...
Cytochrome (Cyt) b559 is a key component of the photosystem II (PSII) complex for its assembly and proper function. Previous studies have suggested that Cytb559 has functional roles in early assembly of PSII and in secondary electron transfer pathways that protect PSII against photoinhibition. In addition, the Cytb559 in various PSII preparations exhibited multiple different redox potential forms. However, the precise functional roles of Cytb559 in PSII remain unclear. Recent site-directed mutagenesis studies combined with functional genomics and biochemical analysis, as well as high-resolution x-ray crystallography and cryo-electron microscopy studies on native, inactive, and assembly intermediates of PSII have provided important new structural and mechanistic insights into the functional roles of Cytb559. This mini-review gives an overview of new exciting results and their significance for understanding the structural and functional roles of Cytb559 in PSII.
... 68,69,72−74 Because the intrinsic intensity of the ν 3 mode for the five-coordinate high-spin complexes is higher than that of the six-coordinate low-spin species, the significantly intense band at 1470 cm −1 could result from the minor five-coordinate species present in the system. 68,69,72,73 Note that the β and α bands (530 and 559 nm) in the Q-band region of the absorption spectrum are characteristic features of bis-Hiscoordinated active sites, observed in neuroglobin, cytoglobin, and cytochrome b type heme proteins. 63,64,77 Hence, the lowspin species can be assigned as a bis-His-coordinated heme center. ...
... Simultaneously, the intensities of both α and β bands at 566 and 535 nm increase ( Figure 5A), consistent with those found in cytochrome b type proteins. 69,72,73,78,79 The weak frequency observed at 1492 cm −1 in the ν 3 region of the rR spectra of the 1:5 heme−Aβ complex indicates that there exists a minor component of the five-coordinate high-spin complex even in the presence of excess Aβ ( Figure S6). Importantly, the low-spin form predominates in the presence of excess Aβ, even under dilute conditions (0.00625 mM; Figure S7). ...
Recent evidence has established the colocalization of amyloid-rich plaques and heme-rich deposits in the human cerebral cortex as a common postmortem feature in Alzheimer's disease (AD). The amyloid β (Aβ) peptides have been shown to bind heme, and the resultant heme-Aβ complexes can generate toxic partially reduced oxygen species (PROS) and exhibit peroxidase activity. The heme-Aβ active site exhibits a concentration-dependent equilibrium between a high-spin mono-His-bound species similar to a peroxidase-type active site and a bis-His-bound six-coordinate low-spin species similar to that of a cytochrome b type active site. The νFe-His (241 cm(-1)) vibration has been identified in the high-spin heme-Aβ active site by resonance Raman spectroscopy. The formation of the low-spin heme-Aβ species is promoted by the His14 and noncoordinating second-sphere Arg5 residues. The high-spin state produces more PROS than the low-spin species. Nonbiological constructs modeling different forms of Aβ (oligomers, fibrils, etc.) suggest that the detrimental high-spin state is likely to dominate under most physiological conditions.
... Both cytochrome protein subunits are of chloroplastic origin (a and b subunits are the products of the psbE and psbF genes, respectively), and span the thylakoid membrane with a single a-helix. Each subunit contains a histidine that coordinates the heme group towards the stromal side of the thylakoid membrane [5,6] making a hexacoordinate low spin iron [7][8][9]. ...
... It is well established that the natural heterodimeric Cyt b 559 is a low spin species with characteristic signals of g z = 3.0-2.9, g y = 2.26, and g x = 1.4 [7,8,31,32]. Membrane preparation with the MBP protein alone was used as control, and showed almost undetectable EPR spectral signals [19]. ...
The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane.
Copyright © 2015 Elsevier B.V. All rights reserved.
... Both cytochrome protein subunits are of chloroplastic origin (a and b subunits are the products of the psbE and psbF genes, respectively), and span the thylakoid membrane with a single a-helix. Each subunit contains a histidine that coordinates the heme group towards the stromal side of the thylakoid membrane [5,6] making a hexacoordinate low spin iron [7][8][9]. ...
... It is well established that the natural heterodimeric Cyt b 559 is a low spin species with characteristic signals of g z = 3.0-2.9, g y = 2.26, and g x = 1.4 [7,8,31,32]. Membrane preparation with the MBP protein alone was used as control, and showed almost undetectable EPR spectral signals [19]. ...
The first step in the photosynthetic reduction of nitrate to ammonia, as carried out by plants and green algae, is catalyzed by the enzyme nitrate reductase. This enzyme serves an assimilatory function in which it catalyzes the exergonic two-electron reduction of nitrate to nitrite with electrons donated by pyridine nucleotides, which are in turn reduced at the end of the photosynthetic electron transport chain by electrons derived originally from water. Although the assimilation of nitrate by photosynthetic organisms appears to be regulated primarily at the nitrate uptake level, the nitrate reductase activity seems to be another key point of control for this pathway. In fact, nitrate reductase from eukaryotic organisms has been demonstrated to exist in two metabolically interconvertible forms, one oxidized/active and the other reduced/inactive.
... Both cytochrome protein subunits are of chloroplastic origin (a and b subunits are the products of the psbE and psbF genes, respectively), and span the thylakoid membrane with a single a-helix. Each subunit contains a histidine that coordinates the heme group towards the stromal side of the thylakoid membrane [5,6] making a hexacoordinate low spin iron [7][8][9]. ...
... It is well established that the natural heterodimeric Cyt b 559 is a low spin species with characteristic signals of g z = 3.0-2.9, g y = 2.26, and g x = 1.4 [7,8,31,32]. Membrane preparation with the MBP protein alone was used as control, and showed almost undetectable EPR spectral signals [19]. ...
The photochemical activity of a prepared zirconium titanate, ZrTiO4, sample has been studied by laser flash photolysis. A short duration laser pulse was used to produce electron–hole pairs; methyl viologen was employed as electron scavenger. The same procedure has been employed to estimate the photochemical activity of two home prepared single oxides, TiO2 (hp) and ZrO2 (hp) and for a commercially available TiO2 (Degussa, P-25). Our results further showed the potential of the laser flash photolysis technique to estimate the photosensitivity of semiconductor oxides and predict a poor photoactivity for the TiO2 (hp) sample in spite of this sample exhibiting (by XRD technique) the anatase as the unique phase.
... One is then led to consider an alternative possibility for the role or function of cytochrome b-559 in the PS II reaction center complex (Fig. 2). A proposed perspective describes the structure of the plastoquinol exit portal in the PSII reaction center (Müh and Zouni 2016;Eerden et al. 2017), through ligation to histidine residues (Babcock et al. 1985), in this case His 23 and His 24, respectively, in the trans-membrane psbE and psbF helices ( Fig. 2A). ...
Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit photosystem II complex which functions in oxygenic photosynthesis, there is presently no consensus on the function of b-559 in the photosynthetic electron transport chain. The inability in earlier times to define a redox-linked function of this cytochrome was, to a large extent, a consequence of an absence of biochemical and structure information to complement an extensive array of spectrophotometric studies of the cytochrome in situ. Based on the location of hetero-dimeric b-559 in the photosystem II reaction center complex, derived from crystal crystallographic structure analysis, and the absence of a necessary redox function for the cytochrome in PSII, it is proposed that the main function of cytochrome b-559 is linked to its role as a structure component in the PSII reaction center complex. This function resides in the association of b-559 through its heme histidine residues in the trans-membrane domains of the PsbE and PsbF subunits of the PSII reaction center. These subunits, along with PsbJ, are inferred, from the analysis of structure, to define the intra-membrane portal in the PSII reaction center for plastoquinol (PQH2) export which, through the PSII complex, provides the redox link to the cytochrome b6f complex in the electron transfer chain.
... Consequently, upon Soret excitation, two ν(C=C) stretching modes are observed in the RR spectra of heme b proteins [72] and their wavenumbers have been found to range from 1618 to 1635 cm −1 . In most heme proteins, two distinct stretching modes have been identified, whereas in a few cases only a single band, deriving from both vinyl groups, has been observed at about 1620 cm −1 (e.g., in Mb, [72] cytochrome b, [73,74] cytochrome c peroxidase (CCP) [70,71] ). In 2003, a direct relationship between the ν(C=C) stretching mode wavenumber and the vinyl group orientations induced by specific protein interactions was established. ...
In heme proteins, the canonical and reversed conformations result from the rotation of the heme group by 180° about the α,γ‐meso axis in the protein pocket. The coexistence of the two different heme orientations has been observed both in proteins reconstituted with hemin and in some native proteins. The reversal of the heme orientation can also change certain functional properties of heme proteins. Complementing the results from other experimental techniques, like circular dichroism and nuclear magnetic resonance, resonance Raman spectroscopy provides detailed information on the structure of the reversed heme. This allows one to elucidate the effects of the heme rotation on the vibrational spectra of the peripheral substituents, especially the vinyl groups. Furthermore, the combination of resonance Raman spectroscopy on single crystals and solution samples of heme proteins is proposed to be a sensitive tool to detect heme orientational disorder, even in the absence of structural data. In heme proteins the canonical and reversed conformations result from the rotation by 180° about the α,γ‐meso axis of the heme group in the protein pocket. This review focuses on the capability and sensitivity of resonance Raman spectroscopy to unravel details of the structure of the reversed heme, elucidating the effects of the heme rotation on the vibrational spectra of the vinyl groups. The discussion is complemented by results from circular dichroism and nuclear magnetic resonance (NMR).
... In intact chloroplasts, the ratio of HP to LP forms was found to be 58 to 31, with respective redox potentials of 383 and 77 mV [242]. In untreated PSII membranes, the ratio HP:IP:LP was estimated as 44:31:25, with redox potentials of 375, 228 and 57 mV, respectively [243,244]. In isolated thylakoid membranes, 85% of cyt b559 was in the HP form [245]. ...
Oxygen is a natural acceptor of electrons in the respiratory pathway of aerobic organisms and in many other biochemical reactions. Aerobic metabolism is always associated with the formation of reactive oxygen species (ROS). ROS may damage biomolecules but are also involved in regulatory functions of photosynthetic organisms. This review presents the main properties of ROS, the formation of ROS in the photosynthetic electron transport chain and in the stroma of chloroplasts, and ROS scavenging systems of thylakoid membrane and stroma. Effects of ROS on the photosynthetic apparatus and their roles in redox signaling are discussed.
... The cytochrome b5 and three of the four hemes of the cytochrome c3 have the imidazole rings of the two coordinated histidines in nearly parallel orientation, while in the cytochrome c3, the imidazole planes are nearly perpendicular. Additionally, there are a number of membrane cytochromes b with bis(histidine) ligands including the two cytochromes b known as the ubiquinone-cytochrome ccytochrome c oxidoreductase and the chloroplast cytochrome b6 [4]. For these iron(III) biological derivatives, there is a correlation between the axial ligand plane orientation and the reduction potentials as reported by Niki et al., [5]. ...
We have successfully synthesized and characterized a new low-spin iron(III) bis(4-cyanopyridine) complex with a meso-porphyrin substituted in the para positions of the phenyls by the methoxy group, namely the bis(4-cyanopyridine)[(meso-tetrakis(4-metoxyphenylporphyrinato)]iron(III) trifluoromethanesulfonate chlorobenzene monosolvate complex with the formula [FeIII(TMPP)(4-CNpy)2]SO3CF3.C6H5Cl (I). This species was characterized through ultraviolet–visible, Fourier-transform infrared and Mössbauer spectroscopy as well as by SQUID magnetometry, cyclic voltammetry, and X-ray crystallography. These characterizations indicated that our synthetic heme model is a low-spin (S = 1/2) coordination compound and especially shows that the structural, electronic and the magnetic properties of complex (I) are closely dominated by the presence of the methoxy σ-donor group at the para positions of the meso-porphyrin.
... Also, the E m value of the HP form is anomalously positive for bis-His ligated heme (for a review, see Ref. [45]). It was suggested that difference between the HP and LP forms is due to a change in mutual orientation of the planes of the His heme ligands [46], however, later estimations showed that the effect would account for only a 50 mV E m shift [47]. Other factors which may affect the redox potential of the heme group in Cyt b559 have been considered such as changes in dielectric properties of the heme environment [48] and different modes of protonation and H-bonding near the heme [32,37,[49][50][51]. ...
Transformation of three-component redox pattern of cytochrome (Cyt) b559 in PS II membrane fragments upon various treatments is manifested in decrease of the relative content (R) of the high potential (HP) redox form of Cyt b559 and concomitant increase in the fractions of the two lower potential forms. Redox titration of Cyt b559 in different types of PS II membrane preparations was performed and revealed that (1) alteration of redox titration curve of Cyt b559 upon treatment of a sample is not specific to the type of treatment; (2) each value of RHP defines the individual shape of the redox titration curve; (3) population of Cyt b559 may exist in several stable forms with multicomponent redox pattern: three types of three-component redox pattern and one type of two-component redox pattern as well as in the form with a single Em; (4) transformation of Cyt b559 proceeds as successive conversion between the stable forms with multicomponent redox pattern; (5) upon harsh treatments, Cyt b559 abruptly converts into the state with a single Em which value is intermediate between the Em values of the two lower potential forms. Analysis of the data using the model of Cyt b559-quinone redox interaction revealed that diminution of RHP in a range from 80 to 10% reflects a shift in redox equilibrium between the heme group of Cyt b559 and the interacting quinone, due to a gradual decrease of 90 mV in Em of the heme group at the virtually unchanged Em of the quinone component.
... The redox potential form of Cyt b 559 is known to be affected by changes in the micro environment around the redox heme (Pospíšil 2011). For example, hydrophobicity around the heme, protonation of His residue, and the axial direction of His imidazole ring are important to determine the redox potential form of Cyt b 559 (Matsuda and Butler 1983;Babcock et al. 1985;Ortega et al. 1988;Krishtalik et al. 1993;Roncel et al. 2001;Pospíšil 2011). Therefore, AA would trigger the conformational changes around Cyt b 559 without disturbing the donor side reaction in PSII. ...
The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b559 (Cyt b559) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b559, which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b559 was oxidized by P680⁺ upon light illumination and re-reduced in the dark, mainly by the electron from the QB site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b559 and induced its reduction under the illumination. This inhibition of Cyt b559 oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b559, rather than that of CEF-PSI in photosynthetic organisms.
... If heme is properly incorporated in b-type cytochromes, the coordination sphere of heme changes due to heme binding to a protein via His or Met residues. This leads to a low-spin, six-fold-coordinated iron in the center of the heme porphyrin ring [63], which becomes evident as a red shift in the SORET-band absorbance maximum compared to free, unbound heme under both, oxidizing and reducing conditions. Furthermore, especially under reducing conditions the SORET-band of b-type cytochromes is sharpened compared to free, unbound heme and the splitting of the α/β band region at 500 to Originally, there is a Cys at this position, which covalently binds a third heme (heme c n ) in vivo. ...
Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α–helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein “halves” for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.
... A role of the hydrogen bond between one of the imidazole nitrogen and an amide carbonyl group of the polypeptide chain has been suggested to cause the conversion between different potential forms of cytochrome b 559 [27]. It seems that the geometry of the axial histidines of the heme-iron [28] as well as its exposure to a more hydrophilic environment are responsible for the variable character of cytochrome b 559 potential forms [29]. ...
Mossbauer spectroscopy was applied, for the first time, to study the interaction of copper ions with the non-heme iron and the heme iron of cytochrome bssg in photosystem II thylakoids isolated from a Chlamydomonas reinhardtii photosystem I minus mutant. We showed that copper ions oxidize the heme iron and change its low spin state into a high spin state. This is probably due to deprotonation of the histidine coordinating the heme. We also found that copper preserves the non-heme iron in a low spin ferrous state, enhancing the covalence of iron bonds as compared to the untreated sample. We suggest that a disruption of hydrogen bonds stabilizing the quinone-iron complex by Cu 2+ is the mechanism responsible for a new arrangement of the binding site of the non-heme iron leading to its more "tense" structure. Such a diamagnetic state of the non-heme iron induced by copper results in a magnetic decoupling of iron from the primary quinone acceptor. These results indicate that Cu does not cause removal of the non-heme iron from its binding site. The observed Cu2+ action on the non-heme iron and cytochrome bsss is similar to that previously observed for α-tocopherol quinone.
... Hydropathy considerations of the two sequences indicate that each polypeptide has only one transmembrane segment. Each segment contains one Downloaded by [187.60.47.52] at 03:19 29 January 2016 histidinc (at residues 23 a and 18 (3) and as there is spectroscopic evidence to suggest that the haem co-ordination must be bis-histidine (Babcock et al., 1985), then it seems likely that the cytochrome has the structure depicted in Figure 17. Although there is some controversy about this in the literature, there is evidence for two cytochrome h-559 per PS2 complex, suggesting that the organization shown in Figure 17 is a monomer of a dimeric arrangement (Cramer. ...
... In addition, there was a change in the relative intensity of the two vinyl modes at 404 and 416 cm −1 and a very large change in relative intensity of the 676 and 687 cm −1 lines, which we attribute to an increased intensity of the 676 cm −1 mode. These are assigned as the heme ν 7 mode originating from heme b and heme c, respectively (18,19). These differences suggest significant conformational changes in the heme b environments. ...
Significance
Many pathogenic bacteria require an aerobic respiratory chain that functions at very low oxygen concentrations. This is accomplished by using a variant of cytochrome c oxidase known as cytochrome cbb 3 . Although related to the human oxidase, cbb 3 has distinct differences that provide opportunities for developing selective drugs. We used site-directed mutagenesis guided and complemented by molecular dynamics simulations to characterize the essential proton channel within the cbb 3 from Vibrio cholerae . Several critical residues are identified, and evidence is presented to show that perturbations near the entrance of the proton channel can have dramatic effects at the active site of the enzyme, more than 25 Å away. This is among the features that distinguish the pathogen respiratory enzyme from the human enzyme.
... Many treatments that modify or remove the Mn cluster are known to shift cyt b 559 from the HP form to the LP form ( [34] and references therein). This potential shift of cyt b 559 involves a small structural rearrangement in the ligand structure around the heme-iron [65] and it is not known how these changes on the lumenal side of the D 1 protein propagate to cyt b 559 which is another protein, >50 ...
We have investigated the electron transfer from reduced tyrosine Y D (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S 2 state multiline EPR signal and the EPR signal from Y D· were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD· signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD·S1 centers, via the intermediate YD·S2 state. The early increase of the S2 state multiline signal involves electron transfer from Y Dred to the S3 state resulting in formation of YD·S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b 559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S 2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the Y DredS2→YD·S1 and YDredS 3→YD·S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed.
... This is similar to what is found for cytochrome b 5 , where the pH-dependent transition is related to deprotonation of one of the axial imidazole ligands or to an imidazole ligand becoming strongly hydrogenbonded to nearby amino-acid residues [284]. It should be noted that the bis-His coordinated heme in cytochrome b 559 in its low-potential or purified form has EPR signals at g z = 2.93 − 2.94, g y = 2.26 − 2.27 and g x = 1.50 − 1.55 [285], which are almost identical to the values obtained for the LS1 of TSCytb. The current observation of the lower g z values for both low-spin ferric forms in TSCytb seems to indicate more relaxed structures for both of the hemes than those in CGCytb or TCytb. ...
Heme proteins of different families were investigated in this work,
using a combination of pulsed and continuous-wave electron paramagnetic
resonance (EPR) spectroscopy, optical absorption spectroscopy, resonance
Raman spectroscopy and laser flash photolysis. The first class of
proteins that were investigated, were the globins. The globin-domain of
the globin-coupled sensor of the bacterium Geobacter sulfurreducens was
studied in detail using different pulsed EPR techniques (HYSCORE and
Mims ENDOR). The results of this pulsed EPR study are compared with the
results of the optical investigation and the crystal structure of the
protein. The second globin, which was studied, is the Protoglobin of
Methanosarcina acetivorans, various mutants of this protein were studied
using laser flash photolysis and Raman spectroscopy to unravel the link
between this protein's unusual structure and its ligand-binding
kinetics. In addition to this, the CN -bound form of this protein was
investigated using EPR and the influence of the strong deformation of
the heme on the unusual low gz values is discussed. Finally, the
neuroglobins of three species of fishes, Danio rerio, Dissostichus
mawsoni and Chaenocephalus aceratus are studied. The influence of the
presence or absence of two cysteine residues in the C-D and D-region of
the protein on the EPR spectrum, and the possible formation of a
disulfide bond is studied. The second group of proteins that were
studied in this thesis belong to the family of the cytochromes. First
the Mouse tumor suppressor cytochrome b561 was studied, the results of
a Raman and EPR investigation are compared to the Human orthologue of
the protein. Secondly, the tonoplast cytochrome b561 of Arabidopsis was
investigated in its natural form and in two double-mutant forms, in
which the heme at the extravesicular side was removed. The results of
this investigation are then compared with two models in literature that
predict the localisation of the hemes in this family of cytochromes.
Finally the preliminary results of a detailed HYSCORE study of the four
hemes in the cytochrome c3 of Desulfovibrio desulfuricans ATCC 27774
are presented.
The cause of phytoplankton blooms has been extensively discussed and largely attributed to favorable external conditions such as nitrogen/phosphorus resources, pH and temperature. Here from the standpoint of hormesis response, we propose that phytoplankton blooms are initiated by stimulatory effects of low concentrations of herbicides as environmental contaminants spread over estuaries and lakes. The experimental results revealed general stimulations by herbicides on Microcystis aeruginosa and Selenastrum capricornutum, with the maximum stimulation in the 30-60% range, depending on the agent and experiment. In parallel with enhancing stimulation, the ratio of HP (high-potential) form to LP (low-potential) form of cytochrome b559 (RHL) was observed decreasing, while intracellular reactive oxygen species (ROS) were observed increasing. We propose that the ROS originated from the thermodynamic transformation of cytochrome b559, enhancing the stimulatory response. Furthermore, the results also proved that thermodynamic states of cytochrome b559 could be modulated by nitric oxide, thus affecting cellular equilibrium of oxidative stress (OS) and correspondingly causing the inhibitory effect of higher concentrations of herbicides on phytoplankton. This suggests that hormesis substantially derives from equilibrium shifting of OS. Moreover, it is reasonable to infer that phytoplankton blooms would be motivated by herbicides or other environmental pollutants. This study provides a new thought into global phytoplankton blooms from a contaminant perspective.
Cytochrome b559 (Cyt b559) is a well-known intrinsic component of the photosystem II (PSH) reaction center (RC) in all oxygen-evolving photosynthetic organisms. Although neither its structure nor its organization in PSII are known, it has been proposed that Cyt b559 is a heme-bridged protein heterodimer with two subunits, a and [3, of 9 and 4 kDa, respectively [1]. An important characteristic of this cytochrome is that it may exhibit different redox potential forms. Usually, Cyt b559 is present in two interconvertible redox forms: a pH-independent high-potential (HP) form (E ‘m = +380 mV) and a pH-dependent low-potential (LP) form (E ’m= +140 mV, pH > 7.6) [2]. The HP form is labile and easily converted to the LP form by a variety of treatments which alter the structure of the membrane.
Cytochrome b559 is one of the ubiquitous constituents of the Photosystem II (PSII) reaction centre. Its presence is a prerequisite for the assembly of PSII, but its function is not understood. In contrast to other cytochromes b which participate in electron transfer reactions, no direct evidence is available to indicate that cytochrome b559 plays a functional role in the primary photosynthetic electron transfer processes [1]. Cytochrome b559 has been shown to assume two different redox-potential forms, a low potential (20 to 80 mV) and a remarkably high potential (330 to 400 mV) form [2]. The two subunits of cytochrome b559, termed α and β, are encoded by the plastid genes psbE and psbF, respectively. The genes are part of the psbE,F,L,J operon in higher plants and cyanobacteria [3,4], while in Chlamydomonas reinhardtii the two genes are found on different DNA strands and thus are transcribed separately [5].
Cytb559 is an integral part of the PSII reaction center. It consists of the membrane spanning α and β subunits [1], both of which are required for the stable assembly and function of PSII [2]. Despite the numerous investigations concerning Cytb559 [for a review see 3] its function, structural arrangement and content (one or two heme group per PSII) is not clear [3]. Cytb559 can exist in several different thermodynamic forms: high-potential (HP. Em≈360 mV) [4], intermediate potential (IP, Em≈230–270 mV) [5], low potential (LP, E.m≈20–80 mV) [4] and very low potential (VLP, Em<45 mV) [6].
Over the last few years it has become increasingly clear that considerable homology exists between the acceptor side components of photosystem II (PS II) and those of reaction centers from purple photosynthetic bacteria (Parson and Ke 1982, Crofts and Wraight 1983, Dutton, Chap. 5, Michel and Deisenhofer, Chap. 8.4, both this Vol.). While one might hardly have expected to see familiar faces among the high potential donors of PS II, recent evidence suggests that some of the same old dogs may have learned new tricks.
Cytochromes are involved in charge-transfer reactions, and many cytochromes contain a transmembrane domain and are part of membrane-localized electron transfer chains. Protoporphyrin IX (heme b) is the first heme product in the tetrapyrrole/heme biosynthesis pathway. In contrast to c-type cytochromes, there is no need for a specialized machinery catalyzing covalent attachment of the heme molecule to a b-type apo-cytochrome, nor is the cofactor further modified, as in a-, d- and o-type cytochromes. Thus, formation of a holo-cytochrome is relatively simple for b-type cytochromes, and this class of proteins probably represents the most ancient members of transmembrane cytochromes. However, assembly of individual transmembrane b-type cytochromes as well as of larger cytochrome complexes involves multiple steps, which have to be tightly controlled and aligned: the apo-protein as well as the heme cofactor needs to be synthesized, targeted to, and integrated into a membrane prior to holo-cytochrome formation. Spontaneous folding and assembly of individual transmembrane b-type cytochromes involves folding of the polypeptide chain and formation of a heme-binding cavity, which allows specific and tight binding of the cofactor. Additional biogenesis steps are eventually required for maturation of transmembrane b-type cytochrome complexes.
In this brief personal perspective, studies, mainly in my laboratory, directed toward understanding of the structure and function of the Fe-metalloprotein electron transport carriers of oxygenic photosynthesis are discussed. This level of understanding and the description of the events that led to this level are inevitably incomplete. The Fe-proteins considered are those, cytochromes b-559 (560), f, b
6, and x, and the Rieske [2Fe−2S] iron-sulfur protein, which have now been placed in a structural context as a result of X-ray diffraction analysis of crystals of Photosystem II and the cytochrome b
6f complex and the Photosystem II reaction center.
In the present paper we describe a new method for the isolation of the 47 kDa protein and the D1-D2-Cyt b559 complex that uses the non-ionic detergent dodecylmaltoside in combination with high concentrations of lithium Perchlorate to dissociate polypeptides of the PSII complex, followed by FPLC ion-exchange chromatography to separate these polypeptides. By carrying out a low temperature EPR study of the two systems we support the proposal that the D1-D2 complex, and not the 47 kDa polypeptide, is the species which binds the reaction center öf PSII. The EPR signal from the spin-polarized triplet was also used as a probe of the stability of the D1-D2-Cyt b559 preparation. It was found that more than 80% of the EPR signal intensity from the spin-polarized triplet could still be observed after incubation of our D1-D2-Cyt b559 complex in the dark at room temperature for five hours.
A summary of biochemical, biophysical, and molecular biological data is presented which led to the identification of two different polypeptides (α and β, MW - 9.16 and 4.27 kDa) in the cytochrome b-559 protein. The presence of a single His residue on each polypeptide, and the conclusion from spectroscopy that the heme coordination must be bis-histidine led to an obligatory requirement for coordination of a single heme through a heme cross-linked dimer. This structure does not have a precedent among soluble or membrane bound cytochromes. The possible participation of the cytochrome in the pathway of photoactivation is discussed.
Heme cross-linking of membrane cytochromes as a structural principle: cytochrome b-559, b
6
, and cytochrome oxidase. After the Mr 9,000 protein of cyt b–559 was purified and its NH2-terminus sequenced (1), its psbE gene and the downstream psbF gene located and sequenced on the spinach chloroplast genome (2), an intermolecular heme cross-linked dimeric structure of cytochrome b–559 was proven by the findings that (a) each polypeptide contains only one histidine (2), (b) the psbE and psbF gene products are present in the purified protein in a 1:1 stoichiometry (3), and (c) the heme coordination is bis-histidine (Fig. la; ref. 4). Thus, a dimer would be required to provide two His residues for heme coordination. There are several reasons, including the 1:1 stoichiometry, for believing that the heme-binding unit is an α-β heterodimer (5), but this has not been rigorously proven.
Photosystem II catalyses the light-driven transfer of electrons from water to plastoquinone, producing oxygen and generating a proton gradient across the thylakoid membrane. The complex may be regarded as made up of three assemblies of polypeptides: a light-harvesting complex (LHCH), a core complex containing the reaction centre and two antenna chlorophyll proteins, and an extrinsic complex concerned with oxygen evolution. Photosystem II is structually the most complex of the supramolecular assemblies of the thylakoid membrane and is currently recognised to be composed of at least 20 different polypeptides.1
Chloroplast cytochrome b-559 is a heme-containing membrane protein intrinsic to the oxygen-evolving Photosystem II core complex (1, 2). Though many theories exist, the actual physiological role of this protein remains enigmatic (3). A 9–10 kDa polypeptide associated with Cyt b-559 has been purified from maize (4) and spinach (5) thylakoid membranes. Antibodies to the HPLC-purified spinach polypeptide, and its 27 amino acid amino-terminal amino acid sequence, were employed to locate the gene, designated psbE, for this polypeptide on the spinach chloroplast genome (6). The psbE gene was completely sequenced and was found to lie between the genes encoding the 51 kDa Photosystem II reaction center chlorophyll-binding protein and apocytochrome f. Moreover, the psbE gene was followed by a 39 codon open reading frame (ORF) transcribed on the same 1.1 – 1.7 Kbp mRNA (6, 7). The polypeptide encoded by this ORF, designated psbF, resembles the psbE polypeptide in containing a single histidine residue within a 25 amino-acid hydrophobic region near the amino terminus. This finding, in combination with electron Chapaumagnetic resonance and Raman spectroscopic data, led to the hypothesis that the heme of Cyt b-559 is coordinated by the two histidines of the psbE and psbF polypeptides in a heterodimeric unit (8, 9).
Cytochrome b-559 in photosystem II reaction center was purified from spinach (Spinacia oleracea L.) and rice (Oryza saliva L.) by a rapid and simple procedure. Their low temperature fluorescence emission and excitation spectra, ultraviolet fluorescence spectra and absolute absorption spectra were presented. The author's purification methods, which enhanced the yield of pure protein and shorted the time for isolation, have several advantages: 1. use of oxygen-evolving PS II core complexes as the starting material in order to avoid disturbing from other cytochromes; 2. isocratic elution of cytochrome b-559 from a DEAE-Sephacel column for eliminating the impurity and yielding the protein in pure state; 3. a simple column procedure for removal of excess Triton X-100. Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. From the results of novel electrophoresis (Tricine-SDS-PAGE), cytochrome b-559 from both spinach and rice reveal two polypeptide bands (apparent molecular weight 9 kD and 4 kD, respectively). By measuring of 77 K fluorescence spectra, it was shown that for the purified cytochrome b-559 there were two excitation peaks at 439 nm and 413 nm, and two emission peaks at 563 nm and 668 nm. This is the first indication that Cyt b-559 is able to emit fluorescence and also transfer excited electrons to chlorophyll. By the use of ultraviolet fluorescence spectra, it was demonstrated for the first time that the location of Trp residue could be in the hydrophobic transmembrane region of cytochrome b-559.
Bovine heart succinate dehydrogenase (SDH) consists of two unlike subunits, namely, flavoprotein (Fp) and iron–sulfur protein (Ip) subunits, with molecular weight averages of 70,000 and 27,000, respectively. This enzyme contains one covalently bound 8α-[W(3)-histidyl]-flavin adenine dinucleotide (FAD) and approximately 8 ± 1 nonheme iron atoms and an equivalent number of acid-labile sulfides (S*). The nonheme iron of SDH was reported to be equally distributed between the Fp and Ip subunits. This chapter describes the newly identified SDH cluster structures that provide the most rational explanations to apparently inconsistent earlier and new experimental data. Based on the most updated knowledge on the amino acid sequence homology, the newly identified structure of three iron–sulfur clusters, and their spin-couplings, the chapter presents a model arrangement of SDH in the mitochondrial membrane. This is the first enzyme to have all three different types of iron–-sulfur clusters within a single polypeptide. All redox-active centers are placed within a distance that allows ready direction of spin–spin interactions.
This chapter discusses genetics and synthesis of chloroplast membrane proteins. Photosystem II appears to be the most complicated of the complexes of the thylakoid membrane, both in structure and in regulation of the synthesis of the individual components. The complex may, superficially, be regarded as made up of three assemblies of polypeptides, a light-harvesting complex (LHC II), a core complex, containing primary electron donor P-680, and an oxygen-evolving complex. Oxygen-evolving PS II preparations may be prepared from thylakoid membranes by detergent fractionation or by mechanical fragmentation followed by partition in an aqueous polymer two-phase system. The polypeptides of the oxygen-evolving complex may be separated from the core complex by a variety of salt treatments. The polypeptides of the light-harvesting complex are most easily resolved by polyacrylamide gel electrophoresis of thylakoid membranes solubilized with sodium or lithium dodecyl sulphate. This produces the green chlorophyll-protein band, CP II, which on staining for protein reveals up to four polypeptides of 24–27 kDa, each of which is believed to bind Chl a and Chl b. In most plants, two polypeptides predominate but other minor polypeptides may be resolved. Furthermore, the genes for the polypeptides of the PS II complexes are distributed between the nuclear and chloroplast genomes in higher plants. The only nuclear genes to be characterized to date are those for the polypeptides of LHC II. Although the mature LflC IT polypeptides are relatively highly conserved, there is considerable variability in the amino acid sequence of the transit peptide.
A complex redox titration pattern of cytochrome (Cyt) b559 in preparations of thylakoid membranes and photosystem (PS) II membrane fragments is commonly attributed to the presence of three conformational forms differing by a structure of the heme microenvironment. However, despite decades of research, structural determinants underlying differences between the redox forms of Cyt b559 have not been defined. In this work, we propose a different interpretation of redox heterogeneity in the native population of Cyt b559 assuming redox interaction between the Cyt b559 heme group and a nearby bound quinone (Q). The interacting quinone is supposed to be plastoquinone QC present in the unusual singly protonated form (QCH). The model successfully explains the unique redox properties of Cyt b559 and may provide a simple and effective mechanism of redox regulation of secondary electron transport in PS II. At the present time, the model of heme-quinone redox interaction can be considered as an alternative to the idea of conformational differences between the native redox forms of Cyt b559.
This chapter discusses the primary reactions of photosystems I and II of algae and higher plants. In photosynthetic organisms, the “primary reactions” fulfil the objective of converting the energy of light into a primary form of chemical energy which lasts for a time compatible with ordinary biochemical processes—that is, milliseconds. In these reactions, a rather large fraction, approximately 40%, of the photon energy is stored as chemical free energy. The primary reactions can be viewed from two major perspectives. Firstly, from a photochemical point of view: pigment molecules are excited to their lowest excited singlet state, which reacts in an electron transfer reaction, the first step of a process of charge separation. Secondly, from a biochemical point of view the reactions take place in highly organized complexes, the reaction centres, which are made up of several classes of molecules that cooperate in fulfilling complementary roles, such as: architectural support, light absorption, energy transfer and electron transfer. All oxygenic organisms, ranging from cyanobacteria to algae and higher plants, contain photosystem I (PS I) and PS II reaction centres, with only minor variations in spite of their large taxonomic and ecological diversity.
Single-crystal electron spin resonance g tensor determinations are reported for three low-spin ferric porphyrin complexes, [Fe(TPP)(py) 2]ClO 4·2THF, K[Fe(TPP)(CN) 2]-2acetone, and Fe(TPP)(CN)(py)·H 2O, all of which exhibit "highly anisotropic low-spin" EPR spectra. These single-crystal measurements allow accurate extraction of the complete g tensor, which in turn provides estimates of the wave functions and relative energies of the three highest occupied molecular orbitals. It is the near-degeneracy of the "d xz" and "d yz" orbitals in these complexes that is responsible for the large g value anisotropy; this near-degeneracy has a number of additional consequences. Analysis of the crystal field parameters, derived from the EPR measurements, indicates that for the (py) 2 and (CN) 2 complexes the product of the three principal g values is negative, whereas for "normal" ferric porphyrin complexes this product is positive. The spin in these complexes is delocalized over the two out-of-plane real d orbitals, whereas in "normal" complexes it is localized in a single real d orbital. For ferric porphyrin complexes with planar axial ligands, this delocalization is associated with a spin-orbit stabilization of the perpendicular ligand conformation. In the case of weak π donors, this stabilization can exceed the crystal field stabilization of the parallel geometry. A crystal structure determination for [Fe(TPP)(py) 2]ClO 4·2THF at 128 K reveals that the axial pyridine ligands adopt a perpendicular conformation that is staggered with respect to the equatorial Fe-N(pyrrole) vectors. The idealized S 4 symmetry about the iron center is consistent with the spectroscopic observations. Crystal data: [Fe(TPP)(py) 2]ClO 4·2THF, space group A2/a, Z = 8, a = 22.814 (2) Å, b = 17.010 (2) Å, c = 27.423 (2) Å, β = 102.51 (1)°, at 128 K.
Cyanophora paradoxa is a flagellated protozoan which possesses unusual, chloroplast-like organelles referred to as cyanelles. The psbE and psbF genes, which encode the two apoprotein subunits of cytochrome b-559, have been cloned from the cyanelle genome of C. paradoxa. The complete nucleotide sequences of these genes and their flanking sequences were determined by the chain-termination, dideoxy method. The psbE gene is composed of 75 codons and predicts a polypeptide of 8462 Da that is seven to nine residues smaller than most other psbE gene products. The psbF gene consists of 43 codons and predicts a polypeptide of 4761 Da. Two open reading frames, whose sequences are highly conserved among cyanobacteria and numerous higher plants, were located in the nucleotide sequence downstream from the psbF gene. The first open reading frame, denoted psbI, is composed of 39 codons, while the second open reading frame, denoted psbJ, is composed of 41 codons. The predicted amino acid sequences of the psbI and psbJ gene products predict proteins of 5473 and 3973 Da respectively. These proteins are probably integral membrane proteins anchored in the membrane by a single, transmembrane alpha helix. The psbEFIJ genes are probably co-transcribed and constitute an operon as found for other organisms. Each of the four genes is preceded by a polypurine sequence which resembles the consensus ribsosome binding sequences for Escherichia coli.
In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385-398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.
The nature of interaction of cytochrome b-559 high potential (HP) with electron transport on the reducing side of photosystem II was investigated by measuring the susceptibility of cytochrome b-559HP to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under different conditions. Submicromolar DCMU concentrations decreased the rate of absorbance change corresponding to cytochrome b-559HP photoreduction while the amplitude was lowered at higher concentrations (up to 10 μM). Appreciable extents of cytochrome b-559HP photoreduction were observed at DCMU concentrations which completely abolished the electron transport from water to methyl viologen under the same experimental conditions. However, the susceptibility of cytochrome b-559HP to DCMU increased with the degree of cytochrome b-559HP oxidation, induced either by ferricyanide or by illumination of low intensity (2 W/m(2)) of red light in the presence of 2 μM carbonyl cyanide-m-chlorophenylhydrazone. Also, the DCMU inhibition was more severe when the pH increased from 6.5 to 8.5, indicating that the unprotonated form of cytochrome b-559HP is more susceptible to DCMU. These results demonstrate that cytochrome b-559HP can accept electrons prior to the QB site, probably via QA although both QA and QB can be involved to various extents in this reaction. We suggest that the redox state and the degree of protonation of cytochrome b-559HP alter its interaction with the reducing side of photosystem II.
Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 μM CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor QA was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.
Cytochrome b559 (Cyt b559) is an intrinsic and essential component of the photosystem II (PSII) reaction center, but its physiological function remains yet undefined. In this study, a partial redox characterization of Cyt b559 in photosynthetic membranes from the transformable unicellular cyanobacterium Synecborystis sp. PCC 6803 (hereafter called Synecborystis 6803) has been performed. In thylakoid membranes extracted by a very mild cell-breaking procedure, as developed in this work, only one redox form of Cyt b559 was found, with a redox potential proper to a low-potential (LP) form. The midpoint redox potential value of this LP form has been shown to be + 170 mV at pH 7.5 in PSII-enriched membranes. In spite of a great variety of cell-breaking treatments used to prepare thylakoid membranes, it has not been possible to detect any high-potential (HP) form of Cyt b559. The absence of this HP form is discussed in terms of its higher lability in Synecborystis 6803 as compared with other cyanobacteria and higher plants. Alternatively, the possibility is considered that a HP form of Cyt b559 does not occur in Synecborystis 6803 because it can not be properly stabilized in the membrane environment.
The amino acid sequences of cytochrome b of complex III from five different mitochondrial sources (human, bovine, mouse, yeast, and Aspergillus nidulans) and the chloroplast cytochrome b6 from spinach show a high degree of homology. Calculation of the distribution of hydrophobic residues with a "hydropathy" function that is conserved in this family of proteins implies that the membrane-folding pattern of the 42-kilodalton (kDa) mitochondrial cytochromes involves 8-9 membrane-spanning domains. The smaller 23-kDa chloroplast cytochrome appears to fold in five spanning domains that are similar to the first five of the mitochondria. Four highly conserved histidines are considered to be the likely ligands for the two hemes. The positions of the histidines along the spanning segments and in a cross section of the membrane-spanning alpha helices implies that two ligand pairs, His-82-His-197/198 and His-96-His-183, bridge the spanning peptides II and V, and the two hemes reside on opposite sides of the hydrophobic membrane core. In addition, the 17-kDa protein of the chloroplast b6-f complex appears to contain one or more of the functions of the COOH-terminal end of the mitochondrial cytochrome b polypeptide.
The cytochromes in spinach chloroplasts were studied using EPR spectroscopy. In addition to the low-spin heme signals previously assigned, cytochrome f (gz 3.51), high-potential cytochrome b-559 (gz 3.08) and cytochrome b-559 converted to a low-potential form (gz 2.94), a high-spin heme signal was induced by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). However, this signal cannot be due to cytochrome b-563 in its native form. The orientation of the cytochromes in the thylakoid membrane was studied in magnetically oriented chloroplasts. Cytochrome b-559 in the native state and in the low-potential form was found to have its heme plane perpendicular to the membrane plane. The orientation was the same for cytochrome b-559 oxidized by low-temperature illumination, which suggests that also the reduced heme is oriented perpendicular to the membrane.
Normal coordinate calculations are carried out for all the in-plane modes of octaethylporphyrinato-Ni (II) and its meso-deuterated and 15N substituted derivatives. With 37 constants of a modified Urey–Bradley force field and a structural model with D4h symmetry, 59 resonance Raman lines (A1g+B1g +A2g+B2g) and 38 infrared bands (Eu) of these three molecules are assigned. The vibrational modes of the Raman active species are represented in terms of the Cartesian atomic displacement vectors. Based on the present results, some important resonance Raman lines of hemoproteins are interpreted. The so-called ’’oxidation state maker’’ (Band IV) is due to an in-phase breathing-like mode of four pyrrole rings although being somewhat deformed by the large contribution of the Cα–N symmetric stretching term. The spin state sensitive Raman lines, namely, Band I and III, are associated mainly with methine bridge (Cα–Cm) stretching modes. Two prominent anomalously-polarized Raman lines of hemoproteins around 1580 and 1300 cm−1 are primarily due to the Cα–Cm stretching and Cm–H bending modes, respectively.
Summary The gene for cytochromeb-559, associated with the photosystem II reaction center, has been located on the spinach plastid chromosome by cell-free
coupled transcription-translation and RNA-programmed hybrid selection translation using appropriate recombinant DNAs, RNA
fractions, and monospecific antisera. The gene is located in the large single-copy segment of the plastid chromosome between
the genes for cytochomef and the P680 chlorophylla apoprotein of photosystem II and transcribed in the opposite direction relative to these genes. The 10 kd protein is decoded
from a bicistronic 1.0 kb mRNA and is apparently not made as a precursor in cell-free rabbit reticulocyte andE. coli lysates.
A rapid and simple procedure is presented for the purification of chloroplast cytochrome b-559. The method is based on the protocol devised by Garewal and Wasserman (Garewal, H.S. and Wasserman, A.R. (1974) Biochemistry 13, 4063–4071), which we have modified to eliminate the requirement for a lengthy electrophoretic step. Novel features of our method include: the use of oxygen-evolving Photosystem II preparations (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539) as the starting material; isocratic elution of cytochrome b-559 from a DEAE-cellulose column (yielding the protein in a pure state); and a simple column procedure for removal of excess Triton X-100. The procedure has been applied to both spinach and maize (Zea mays L.). Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. Lithium dodecyl sulfate polyacrylamide gel electrophoresis of cytochrome b-559 from both spinach and maize reveals a major polypeptide band (apparent molecular mass = 9 kDa), and two minor bands (apparent molecular masses = 10 kDa and 6 kDa).
Cytochrome b5, with a molecular weight of about 1.2 · 104 was highly purified from pig liver.The purified oxidized cytochrome b5 was investigated by the following three methods: 1.(I) Absorption spectrophotometry at 23 °C and 77 °K.2.(II) Electron Paramagnetic Resonance (EPR) spectroscopy at 20 °K.3.(III) Kinetic measurements of the reaction with CN− in the temperature range from 22.25 to 46.75 °C. I and IIhhave demonstrated that: 3.1.1. In the pH region from pH 5.0 to 11.0, oxidized cytochrome b5 is in a purely low-spin state between 23 °C and 20 °K.3.2.2. Below pH 4.0, the spin state reversibly changes to high-spin between 23 °C and 20 °K. This high-spin state is found to be due to the hemin released from cytochrome b5.3.3.3. Above pH 12.0, the spin state reversibly changes to another type of low-spin state between 23 °C and 20 °K, which is thought to come from a distorted protein structure but not from the liganding of OH−.3.4.4. Energy for three t2g orbitals calculated in one hole formalism shows a high symmetry of ligand coordination for the low-spin state at pH 6.2 and a lowering of the symmetry for another type of low-spin state at pH 12.0. III has demonstrated that3.5.5. The reaction with CN− is bi-phasic. The fast reaction in the cytochrome b5 monocyanide complex formation, and the slow one is the hemin dicyanide complex formation.3.6.6. The activation energy for fast and slow reactions are both 25.1 kcal mole. The values of entropy of activation for fast and slow reactions are 12.1 and 11.5 entropy units, respectively. The protein structure of cytochrome b5 in comparison with that of cytochrome c based on the results above as well as those of X-ray studies by Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, O. B., Samoson, L., Cooper, A. and Margoliash, E. (1971) J. Biol. Chem. 246, 1511–1535 and Mathews, F. S., Levine, M. and Argos, P. (1972) J. Mol. Biol. 64, 449–464 are discussed.
The effect of a liposome environment on cytochrome b-559 was examined in two types of Photosystem II particles. A substantial fraction of the low-potential cytochrome b-559 of Photosystem II core particles, which do not evolve oxygen, was restored to a high-potential form in the liposome preparation. A preparation of oxygen-evolving Photosystem II particles, which was selected on the basis of having a relatively low rate of oxygen evolution (68 μmol oxygen/mg chlorophyll per h), showed very little high-potential cytochrome b-559 and a less-than-normal amount of variable-yield fluorescence. In the liposome preparation, however, these particles showed considerably more high-potential cytochrome b-559, an almost-normal amount of variable-yield fluorescence and a substantially greater rate of oxygen evolution (183 μmol oxygen/mg chlorophyll per h).
A simple method is presented for calculating the parameters of the hole model for distorted octahedral, low spin (τ2g)5 complexes. In the case of negligible covalent bonding explicit formulas for the coefficients of the Kramer's doublet, ±a|ξ ± > — ib | η ± > — c | ζ ∓ >, are a (gz + gy)/4p, b (gz − gx)/4p, c (gy − gx)/4p, where gz + gy − gx 2p2 is inherently positive for all correct choices of sign for the principal g values. The two numerically largest g values define the plane and orientation of the orbital with the largest coefficient, which in turn indicates the directions of maximal unpaired spin density. The energy of η with respect to ξ (in units of λ, the spin-orbit coupling constant) is A gx/(gz + gy) + gy/(gz — gx), and of ζ is B gx/(gz + gy) + gz/(gy − gx). The tetragonal splitting, (), and the rhombic, . For a proper axis system, where z is the tetragonal axis, . The product gzgygx, independent of axes, and positive for free electrons, is shown to be positive for tetragonal and negative for nearly octahedral complexes. It is considered positive for hemes. In this method coefficients will only be normalized when there is no covalency. For the majority of published cases they are, to about 1%. Since this discrepancy is larger than can be caused by propagated errors, covalency must be the rule. For comparative purposes A and B, uncorrected for covalency, should still be useful. Examination of published complete g tensors for five hemes shows that the largest g value is nearly normal to the heme plane. If the g values are taken positive and labelled so that gz>gy>gx, then the proper tetragonal axis is roughly normal to plane of the ring in hemes, but not in chlorins.
Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O2 evolution. The preparation contains four managanese, one cytochrome b-559, one Signal IIf and one Signal IIs per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z+ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.
Analysis of a 0.6 kb fragment of the spinach plastic chromosome adjacent to the 3' end of the apocytochrome f gene has disclosed two uninterrupted reading frames of 83 and 39 triplets, the product of the first one being apocytochrome b-559. The first 27 predicted amino acid residues had been verified by protein sequence analysis and the molecular mass of 9390 Da derived from the amino acid sequence deduced here is close to that of the authentic protein. The two genes are transcribed by a bicistronic RNA in a direction opposite to that of cytochrome f, and their translation stop/potential ribosome binding sites overlap. Features of the two genes resembling those of bacterial genes include putative tetra- or pentanucleotide ShineDalgarno sequences, Pribnow boxes, ‘−35’ promotor consensus sequences and possibly a transcription termination region. Both gene structure and products of DNA- or RNA-programmed cell-free translation preclude that apocytochrome b-559 is made as a precursor. The amino acid sequence includes only one histidine residue located in a predicted secondary structure of strong hydrophobicity which indicates the intriguing possibility that more than one protein chain must cooperate in heme binding of this cytochrome.
We have observed an anisotropic EPR spectrum in a frozen solution of the low-spin (S = 12;) hemoprotein ferricytochrome c. These results are interpreted in terms of the theory for the low-spin ferric ion in an octahedral crystal field which is subject to an (axial) distortion along the axis perpendicular to the heme plane and a (rhombic) distortion in the plane of the heme, and a spin-orbit constant which is less than that found in the free ion. This model also leads to calculated values for the effective magnetic moment which agree well with experiment.
1. The midpoint potential of cytochrome b6 in freshly prepared coupled chloroplasts is approximately +5 mV and the slope of the titration curve is characteristic of a two-electron transition. Subsequent titrations performed one-half hour or more after chloroplast preparation show a slight negative shift of the midpoint potential and a one-electron slope.2. Titrations of cytochrome b6 in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) or NH4Cl show the midpoint potential of approximately half the b6 complement negatively shifted to about −140 mV.3. Brief exposure of the chloroplasts to NH4Cl and actinic illumination under typical uncoupling conditions with controlled potential conditions causes a shift in oxidation state consistent with a lowering of the average midpoint potential to about −100 mV.It is concluded that the midpoint potential of at least half the cytochrome b6 undergoes a negatively directed shift of 100–150 mV under uncoupling conditions in the presence of NH4Cl and FCCP.
Laser lines in resonance with the Soret band optical transitions of several heme proteins and heme model compounds have been used to obtain Raman spectra of these species. Correlations between the observed frequency of a polarized mode in the 1560-1600-cm-1 region and the heme iron spin and coordination geometry have been developed. The position of this band is also a function of the pattern of porphyrin pyrrole ring beta-carbon substitution, and therefore structural information can be extracted from the Raman data only after this dependence has been taken into account. Quantitative correlations between the frequency of this band and the porphyrin core size are presented for three commonly occurring classes of heme compounds: (a) protoheme derivatives, (b) iron porphyrins in which all ring positions are saturated, and (c) heme alpha species. A polarized mode in the 1470-1510-cm-1 region is also consistently enhanced upon Soret excitation of these compounds, but is relatively insensitive to peripheral substituents, and can be used in conjunction with the polarized mode described above to assign heme geometries. In the frequency region above 1600 cm-1, a vibration is observed which also responds to changes in porphyrin geometry. However, this band is sometimes obscured by vibrations of unsaturated beta-carbon substituents, particularly in the case of protoheme derivatives. The normal coordinate analysis developed by Abe and co-workers [Abe, M., Kitagawa, T., & Kyogoku, Y. (1978) J Chem. Phys. 69, 4526-4534] is used to rationalize the dependence of the various modes on porphyrin geometry and peripheral substitution.
The oxidation-reduction potential of purified cytochrome b has been determined. At pH 7.0 the E′0 is −0.34 V. This potential can be raised significantly by formation of a complex between the cytochrome b and mitochondrial structural protein. The cytochrome b-structural protein complex is a monomeric species, soluble at neutral pH. Reduction of the cytochrome enhances the rate and extent of formation of the complex. The implications of these observations are discussed.