CW-EPR and ENDOR study of cytochrome c6 from Anabaena PCC 7119

The detailed analysis of the continuous-wave electron paramagnetic resonance and electron nuclear double resonance measurements on cytochrome c(6) from Anabaena PCC7119 reveals several electronic and structural properties of this hemeprotein. The oxidized protein shows two forms that differ in the arrangement of the residues that act as heme axial ligands. Information about the orientation of these residues is obtained for one of the forms, which turns out to differ from that found in the reduced protein from x-ray experiments. The biological significance of these results is discussed.     

Calcium ligation in photosystem II under inhibiting conditions

In oxygenic photosynthesis, PSII carries out the oxidation of water and reduction of plastoquinone. The product of water oxidation is molecular oxygen. The water splitting complex is located on the lumenal side of the PSII reaction center and contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen from water; the five sequentially oxidized forms of the water splitting complex are known as the Sn states, where n refers to the number of oxidizing equivalents stored. Calcium plays a role in water oxidation; removal of calcium is associated with an inhibition of the S state cycle. Although calcium can be replaced by other cations in vitro, only strontium maintains activity, and the steady-state rate of oxygen evolution is decreased in strontium-reconstituted PSII. In this article, we study the role of calcium in PSII that is limited in water content. We report that strontium substitution or 18OH2 exchange causes conformational changes in the calcium ligation shell. The conformational change is detected because of a perturbation to calcium ligation during the S1 to S2 and S2 to S3 transition under water-limited conditions.     

H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin

The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN⁻ have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors. Ligand binding in heme-containing proteins is determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. Since both the RR Fe–OH⁻ and Fe–CN⁻ frequencies are very sensitive to the distal environment, detailed information on structural variations has been obtained. The hydroxyl ligand binds only the WT protein giving rise to two different conformers. In form 1 the anion is stabilized by H-bonds with TrpG8, TyrCD1 and a water molecule, in turn H-bonded to TyrB10. In form 2, H-bonding with TyrCD1 is mediated by a water molecule. Unlike the OH⁻ ligand, CN⁻ binds both WT and the triple mutant giving rise to two forms with similar spectroscopic characteristics. The overall results clearly indicate that H-bonding interactions both with distal residues and water molecules are important structural determinants in the active site of Tf-trHb. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Ammonia displaces methanol bound to the water oxidizing complex of photosystem II in the S2 state

Ammonia and methanol both bind to the water oxidising complex of photosystem II during its turnover, possibly at sites where water binds during the normal water oxidation process. We have investigated the interaction between these two water analogues at the S2 state of the water oxidising cycle using electron magnetic resonance techniques. We find evidence that ammonia displaces methanol from its binding site.     

Mitochondrial dysfunction in patients with severe sepsis: An EPR interrogation of individual respiratory chain components

Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron-sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ^·-SQ^·). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219-223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ^·-SQ^·) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron-sulfur redox centres is linked to mortality.     

Calcium exchange and structural changes during the photosynthetic oxygen evolving cycle

PSII catalyzes the oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. PSII contains an oxygen-evolving complex, which is located on the lumenal side of the PSII reaction center and which contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen. This process produces five Sn-states, where n refers to the number of oxidizing equivalents stored. Calcium is required for oxygen production. Strontium is the only divalent cation that replaces calcium and maintains activity. In our previous FT-IR work, we assessed the effect of strontium substitution on substrate-limited PSII preparations, which were inhibited at the S3 to S0 transition. In this work, we report reaction-induced FT-IR studies of hydrated PSII preparations, which undergo the full S-state cycle. The observed difference FT-IR spectra reflect long-lived photoinduced conformational changes in the oxygen-evolving complex; strontium exchange identifies vibrational bands sensitive to substitutions at the calcium site. During the S1' to S2' transition, the data are consistent with an electrostatic or structural perturbation of the calcium site. During the S3' to S0' and S0' to S1' transitions, the data are consistent with a perturbation of a hydrogen bonding network, which contains calcium, water, and peptide carbonyl groups. To explain our data, persistent shifts in divalent cation coordination must occur when strontium is substituted for calcium. A modified S-state model is proposed to explain these results and results in the literature.     

Bidirectional electron transfer in photosystem I: Replacement of the symmetry-breaking tryptophan close to the PsaB-bound phylloquinone (A1B) with a glycine residue alters the redox properties of A1B and blocks forward electron transfer at cryogenic temperatures

A conserved tryptophan residue located between the A_1B and F_X redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A_1A and the A_1B phyllo(semi)quinones. The kinetics of A₁⁻ reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F_X is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F_X leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F_A and F_B by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A_1B.     

Protein-cofactor interactions in bioenergetic complexes: The role of the A1A and A1B phylloquinones in Photosystem I

This review focuses on phylloquinone as an indispensable link between light-induced charge separation and subsequent charge stabilization in Photosystem I (PS I). Here, the role of the polypeptide in conferring the necessary kinetic and thermodynamic properties to phylloquinone so as to specify its functional role in PS I electron transfer is discussed. Photosynthetic electron transfer and the role of quinones in Type I and Type II reaction centers are introduced at the outset with particular emphasis on the determination of redox potentials of the cofactors. Currently used methodologies, particularly time-resolved optical spectroscopy and varieties of magnetic resonance spectroscopy that have become invaluable in uncovering the details of phylloquinone function are described in depth. Recent studies on the selective alteration of the protein environment and on the incorporation of foreign quinones either by chemical or genetic means are explored to assess how these studies have improved our understanding of protein-quinone interactions. Particular attention is paid to the function of the H-bond, methyl group and phytyl tail of the phylloquinone in interacting with the protein environment.     

Two-dimensional pulsed electron spin resonance characterization of N-labeled archaeal Rieske-type ferredoxin

Two-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly ¹⁵N-labeled archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 has been conducted in comparison with the previously characterized high-potential protein homologs. Major differences among these proteins were found in the hyperfine sublevel correlation (HYSCORE) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinated) peptide nitrogens near the reduced clusters. They are less pronounced in the HYSCORE spectra of ARF than those of the high-potential protein homologs, and may account for the tuning of Rieske-type clusters in various redox systems.     

Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O(2) or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.     

Location of the extrinsic subunit PsbP in photosystem II studied by pulsed electron-electron double resonance

The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (Y_D) in PsbD, the D2 subunit of plant photosystem II, were 50.8?±?3.5?Å, 54.9?±?4.0?Å, 57.8?±?4.9?Å, and 58.4?±?14.1?Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of Y_D relative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed Y_D coordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca²⁺-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y_Z. The ratio of the intensities between the g=2 broad signal and the Y_Z signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y_Z signal decayed together in parallel with the appearance of the S₂ state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S₁X state in the transition from the S₁ to the S₂ state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y_Z. Possible reactions in the S state transitions in Ca²⁺-depleted PS II were discussed.     

Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: Characterization of the rbcL deletion mutant of tobacco

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The ΔrbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q_A and Q_B, whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of ΔrbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F₀ ‘dark rise’ in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O₂ in ΔrbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the ΔrbcL mutant under growth conditions. This protective capacity was rapidly exceeded in ΔrbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.     

Structural characterization of ferric hemoglobins from three antarctic fish species of the suborder notothenioidei

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.     

Models for coordination site of cytochrome P-450, characterization of hemin-thiolato complexes with S, O, and N donor ligands by electronic absorption and electron spin resonance spectra

Bisthiolato-hemin complexes exhibiting "two split Soret bands" at 370 and 460 nm, classified into "hyperporphyrin spectrum" was prepared with naturally occurring porphyrins (Fe(III)protoporphyrin IX and its dimethyl ester), thioglycolate esters, and tetramethylammonium hydroxide in organic solvents. The structure of the complexes was characterized by electronic absorption and electron spin resonance (ESR) spectrometries. These complexes were stable under air at room temperature, their apparent half-lives being about 30 min monitored by the intensities of the two Soret bands. Thus the bisthiolato-hemin complex containing thioglycolate ester was shown to be a model for the cytochrome P450(P450)-thiolato binding complex. Ligand exchange reactions of the bisthiolato-hemin complex with imidazole or methanol indicated that the intermediate species are stabilized as thiolato-hemin-imidazole or -methanol complexes. The latter intermediate complex was suggested to be a good model for low-spin ferric P450 as characterized by distinct beta- and alpha-bands at 530 and 560 nm, respectively, as well as a single Soret peak at approximately 410 nm. The result of the analysis on ESR g values and crystal field parameters for the bisthiolato-hemin, thiolato-hemin-imidazole, and thiolato-hemin-oxygen ligand complexes comparing with those for P450 itself and the ligand binding complexes revealed that the sixth ligand trans to the fifth thiolato ligand of the low-spin ferric P450 can be an oxygen atom of water molecule.     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.     

Structure of Self-Aggregated Alamethicin in ePC Membranes Detected by Pulsed Electron-Electron Double Resonance and Electron Spin Echo Envelope Modulation Spectroscopies

PELDOR spectroscopy was exploited to study the self-assembled super-structure of the [Glu(OMe)^7,18,19]alamethicin molecules in vesicular membranes at peptide to lipid molar ratios in the range of 1:70-1:200. The peptide molecules were site-specifically labeled with TOAC electron spins. From the magnetic dipole-dipole interaction between the nitroxides of the monolabeled constituents and the PELDOR decay patterns measured at 77 K, intermolecular-distance distribution functions were obtained and the number of aggregated molecules (n ≈ 4) was estimated. The distance distribution functions exhibit a similar maximum at 2.3 nm. In contrast to Alm16, for Alm1 and Alm8 additional maxima were recorded at 3.2 and ∼5.2 nm. From ESEEM experiments and based on the membrane polarity profiles, the penetration depths of the different spin-labeled positions into the membrane were qualitatively estimated. It was found that the water accessibility of the spin-labels follows the order TOAC-1 > TOAC-8 ≈ TOAC-16. The geometric data obtained are discussed in terms of a penknife molecular model. At least two peptide chains are aligned parallel and eight ester groups of the polar Glu(OMe)^18,19 residues are suggested to stabilize the self-aggregate superstructure.     

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

Intramembrane Water Associated with TOAC Spin-Labeled Alamethicin: Electron Spin-Echo Envelope Modulation by D2O

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D₂O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D₂O-ESEEM intensities decrease from TOAC¹ to TOAC⁸ and TOAC¹⁶ but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC¹⁶ is located in the bilayer leaflet opposite to that of TOAC¹ and TOAC⁸. Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D₂O-ESEEM intensity is greatest for TOAC⁸, indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.