The heme environment of mouse neuroglobin: histidine imidazole plane orientations obtained from solution NMR and EPR spectroscopy as compared with X-ray crystallography

The ¹H NMR chemical shifts of the heme methyl groups of the ferriheme complex of metneuroglobin (Du et al. in J. Am. Chem. Soc. 125:8080–8081, 2003) predict orientations of the axial histidine ligands (Shokhirev and Walker in J. Biol. Inorg. Chem. 3:581–594, 1998) that are not consistent with the X-ray data (Vallone et al. in Proteins Struct. Funct. Bioinf. 56:85–94, 2004), and the EPR spectrum (Vinck et al. in J. Am. Chem. Soc. 126:4516–4517, 2004) is only marginally consistent with these data. The reasons for these inconsistencies appear to be rooted in the high degree of aqueous solution exposure of the heme group and the fact that there are no strong hydrogen-bond acceptors for the histidine imidazole N–H protons provided by the protein. Similar inconsistencies may exist for other water-soluble heme proteins, and ¹H NMR spectroscopy provides a simple means to verify whether the solution structure of the heme center is the same as or different from that in the crystalline state.     

Detection of heme oxygenase activity in a library of four-helix bundle proteins: towards the de novo synthesis of functional heme proteins

Design and chemical synthesis of de novo heme proteins with enzymatic activity on cellulose membranes is described. 352 antiparallel four-helix bundle proteins with a single histidine for heme ligation were assembled from three different sets of short amphipathic helices on membrane-bound peptide templates. The templates were coupled by linkers to cellulose membranes of microplate format, which could be cleaved for control of intermediate and final products. The incorporation of heme and the heme oxygenase activity of the 352 proteins were monitored by measuring UV-visible spectra directly on the cellulose. The kinetics of the heme oxygenase reaction was studied by monitoring the decrease of the Soret band and the transient absorbance of verdoheme being an intermediate product in the formation of biliverdin. Four of the proteins covering a broad range of the enzymatic rate constants were selected and synthesized in solution for further characterization. Detailed studies by redox potentiometry, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy yielded information about the aggregation state of the proteins, the spin state and the putative coordination environment of the iron. The amount of five-coordinated high-spin iron and a positive reduction potential were found to promote the oxygenase activity of the proteins.     

When an amide is more like histidine than imidazole: the role of axial ligands in heme catalysis

 Of the many subtle protein-cofactor interactions which facilitate oxidative catalysis by heme enzymes, the role of the axial ligand has for some time appeared to be fairly well understood. Recent studies from several laboratories, however, have provided good reason to reemphasize the importance of secondary interactions between the axial ligand and protein, as the results suggest that simple ligand identity is neither necessary nor sufficient for function. It has been widely proposed that the strong hydrogen bond between a proximal carboxylate and the histidine ligand of peroxidases assists O–O bond heterolysis and stabilizes the Fe(IV)=O center that is produced. Recent replacements of the axial ligand in a number of heme proteins have produced a few surprises, suggesting that the subtle interactions between the ligand and protein may in some cases be more important than the actual identity of the ligand. Received and accepted: 7 May 1996     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH₃CH(OH), CO₂, O₂, and e⁻_aq′ and the oxidation of the reduced cytochrome c derivatives by Fe(CN)³⁻₆ were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partitioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

Studies of the orientation of the mitochondrial redox carriers III. Orientation of the gx and gy axes of the hemes of cytochrome oxidase with respect to the plane of the membrane in oriented membrane multilayers

The EPR absorption properties of the hemes of cytochrome oxidase and their liganded derivatives were examined in oriented multilayers from isolated oxidase, mitochondrial membranes and membrane fragments of a bacterium, Paracoccus denitrificans. The hemes of the oxidase in all the systems investigated were oriented normal to the plane of the multilayers. The directions of the g signals corresponding to the g_x and g_y axes of the g tensor were found to be different in low-spin ferric heme in fully oxidized oxidase and in half-reduced liganded oxidase. It is suggested that this different orientation of g_x and g_y in fully oxidized oxidase and half-reduced liganded oxidase arises because the respective EPR signals belong to two different hemes, those of cytochrome a and a₃.     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.     

Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic ¹³C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.     

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV–vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately −500 mV (vs. the standard hydrogen electrode). Across this potential window the UV–vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.     

Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (azurin, cytochrome c, myoglobin) and the bacterial photosynthetic reaction center reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. The β-sheet azurin structure efficiently and isotropically mediates coupling with an exponential distance-decay constant of 1.1?Å^–1. The experimentally derived distance-decay constant of 1.4?Å^–1 for long-range ET in myoglobin and the reaction center suggests that hydrogen-bond couplings are weaker through α helices than across β sheets. The donor-acceptor interactions of systems with comparable tunneling energies fall into two coupling zones: the β zone (bounded by distance-decay constants of 0.9?and 1.15 Å^–1) includes all the β-sheet (azurin) couplings and all but one coupling in cytochrome c; the α zone (boundaries: 1.25 and 1.6?Å^–1) includes less strongly coupled donor-acceptor pairs in myoglobin and the reaction center as well as a relatively weakly coupled pair in cytochrome c.     

Specific Function of the Met-Tyr-Trp Adduct Radical and Residues Arg-418 and Asp-137 in the Atypical Catalase Reaction of Catalase-Peroxidase KatG

Catalase activity of the dual-function heme enzyme catalase-peroxidase (KatG) depends on several structural elements, including a unique adduct formed from covalently linked side chains of three conserved amino acids (Met-255, Tyr-229, and Trp-107, Mycobacterium tuberculosis KatG numbering) (MYW). Mutagenesis, electron paramagnetic resonance, and optical stopped-flow experiments, along with calculations using density functional theory (DFT) methods revealed the basis of the requirement for a radical on the MYW-adduct, for oxyferrous heme, and for conserved residues Arg-418 and Asp-137 in the rapid catalase reaction. The participation of an oxyferrous heme intermediate (dioxyheme) throughout the pH range of catalase activity is suggested from our finding that carbon monoxide inhibits the activity at both acidic and alkaline pH. In the presence of H₂O₂, the MYW-adduct radical is formed normally in KatG[D137S] but this mutant is defective in forming dioxyheme and lacks catalase activity. KatG[R418L] is also catalase deficient but exhibits normal formation of the adduct radical and dioxyheme. Both mutants exhibit a coincidence between MYW-adduct radical persistence and H₂O₂ consumption as a function of time, and enhanced subunit oligomerization during turnover, suggesting that the two mutations disrupting catalase turnover allow increased migration of the MYW-adduct radical to protein surface residues. DFT calculations showed that an interaction between the side chain of residue Arg-418 and Tyr-229 in the MYW-adduct radical favors reaction of the radical with the adjacent dioxyheme intermediate present throughout turnover in WT KatG. Release of molecular oxygen and regeneration of resting enzyme are thereby catalyzed in the last step of a proposed catalase reaction.     

Heat-induced changes in the EPR signal of tyrosine D ($$ Y^{{OX}}_{D} $$): a possible role of Cytochrome b559

The present study for the first time describes a close relationship between a change in the states of Cyt b559, a damage to Mn complex and a rapid reduction of tyrosine D (Y_D) as a function of temperature in spinach thylakoid membranes. Measurements of the EPR signal of dark stable tyrosine D in heat-treated thylakoid membranes showed a gradual decay of the oxidized state of tyrosine D with the progression of temperature. Simultaneously, it leads to the conversion of high-potential Cytochrome b559 into its low-potential form. We have speculated a possible involvement of Cytochrome b559 in the primary reduction events of tyrosine D in dark at high temperature. However, rapid reduction of tyrosine D may also be due to the disassembly of the Mn clock, which causes exposure of Y_D to the lumen and thereby its reduction by some unknown factor. These conclusions are supported by the measurements of Mn²⁺ release and thermoluminescence curves of various charge pairs in heat-treated thylakoid membranes. The results reveal an important aspect on the role of Cyt b559 in PS II during temperature stress.     

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa₃ (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a₃, and time-resolved experiments indicate that even transient binding to Cu_B does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to Cu_B. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species.     

A spectroscopic and voltammetric study of the pH-dependent Cu(II) coordination to the peptide GGGTH: relevance to the fifth Cu(II) site in the prion protein

The GGGTH sequence has been proposed to be the minimal sequence involved in the binding of a fifth Cu(II) ion in addition to the octarepeat region of the prion protein (PrP) which binds four Cu(II) ions. Coordination of Cu(II) by the N- and C-protected Ac-GGGTH-NH(2) pentapeptide (P(5)) was investigated by using potentiometric titration, electrospray ionization mass spectrometry, UV-vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry experiments. Four different Cu(II) complexes were identified and characterized as a function of pH. The Cu(II) binding mode switches from NO(3) to N(4) for pH values ranging from 6.0 to 10.0. Quasi-reversible reduction of the [Cu(II)(P(5))H(-2)] complex formed at pH 6.7 occurs at E (1/2)=0.04 V versus Ag/AgCl, whereas reversible oxidation of the [Cu(II)(P(5))H(-3)](-) complex formed at pH 10.0 occurs at E (1/2)=0.66 V versus Ag/AgCl. Comparison of our EPR data with those of the rSHaPrP(90-231) (Burns et al. in Biochemistry 42:6794-6803, 2003) strongly suggests an N(3)O binding mode at physiological pH for the fifth Cu(II) site in the protein.     

Folding of the prion peptide GGGTHSQW around the copper(II) ion: identifying the oxygen donor ligand at neutral pH and probing the proximity of the tryptophan residue to the copper ion

The GGGTHSQW sequence in the amyloidogenic part of the prion protein is a potential binding site for Cu(II). We have previously studied the binding of copper to the shorter GGGTH peptide and showed that it is highly pH dependent (Hureau et al. in J. Biol. Inorg. Chem. 11:735–744, 2006). Two predominant complexes could be characterized at pH 6.7 and 9.0 with equatorial binding modes of 3N1O and 4N for the metal ion, respectively. In this work, we have further investigated the coordination of Cu(II) to the GGGTH peptide as well as the longer GGGTHSQW peptide in order to identify the oxygen donor ligand at neutral pH and to study the proximity and redox activity of the tryptophan residue of the latter. The results for both peptides show that, at pH 6.7, Cu(II) is coordinated by a carbonyl peptide backbone. At higher pH values, the carbonyl ligand dissociates and the coordination changes to a 4N binding mode, inducing a structural rearrangement that brings the GGGTHSQW peptide’s tryptophan residue into the vicinity of the copper ion, thus affecting their respective redox properties.     

Frost hardening and photosynthetic performance of Scots pine (Pinus sylvestris L.). II. Seasonal changes in the fluidity of thylakoid membranes

The fluidity of chloroplast thylakoid membranes of frost-tolerant and frost-sensitive needles of␣three- to four-year-old Scots pine (Pinus sylvestris L.) trees, of liposomes produced from the lipids of the thylakoids of these needles, and of liposomes containing varying amounts of light-harvesting complex (LHC) II protein was investigated by means of electron paramagnetic resonance (EPR) measurements using spin-labelled fatty acids as probes. Broadening of the EPR-resonance signals of 16-doxyl stearic acid in chloroplast membranes of frost-sensitive needles and changes in the amplitudes of the peaks were observed upon a decrease in temperature from +30 °C to −10 °C, indicating a drastic loss in rotational mobility. The lipid molecules of the thylakoid membranes of frost-tolerant needles exhibited greater mobility. Moderate frost resistance could be induced in Scots pine needles by short-day treatment (Vogg et al., 1997, Planta, this issue), and growth of the trees under short-day illumination (9 h) resulted in a higher mobility of the chloroplast membrane lipids than did growth under long-day conditions (16 h). The EPR spectrum of thylakoids from frost-tolerant needles at −10 °C was typical of a spin label in highly fluid surroundings. However, an additional peak in the low-field range appeared in the subzero temperature range for the chloroplast membranes of frost-sensitive needles, which represents spin-label molecules in a motionally restricted surrounding. The EPR spectra of thylakoids and of liposomes of thylakoid lipids from frost-hardy needles were identical at +30 °C and −10 °C. The corresponding spectra from frost-sensitive plants revealed an additional peak for the thylakoids, but not for the pure liposomes. Hence, the domains with restricted mobility could be attributed to protein-lipid interactions in the membranes. Broadening of the spectrum and the appearance of an additional peak was observed with liposomes of pure distearoyl phosphatidyl glycerol modified to contain increasing amounts of LHC II. These results are discussed with respect to a loss of chlorophyll and chlorophyll-binding proteins in thylakoids of Scots pine needles under winter conditions. Received: 3 March 1997 / Accepted: 16 July 1997     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: II. KINETICS AND MECHANISM OF BINDING*

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c₁ of detergent-solubilized bc₁ complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c₁-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mm Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mm, the rate leveled off, indicating a rate-limiting conformational step with lifetime ∼1 s; and (iii) at Im concentrations above 100 mm, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.     

Nitrogen ligation to the manganese cluster of Photosystem II in the absence of the extrinsic proteins and as a function of pH

Three extrinsic proteins (PsbO, PsbP and PsbQ), with apparent molecular weights of 33, 23 and 17 kDa, bind to the lumenal side of Photosystem II (PS II) and stabilize the manganese, calcium and chloride cofactors of the oxygen evolving complex (OEC). The effect of these proteins on the structure of the tetramanganese cluster, especially their possible involvement in manganese ligation, is investigated in this study by measuring the reported histidine-manganese coupling [Tang et al. (1994) Proc Natl Acad Sci USA 91: 704–708] of PS II membranes depleted of none, two or three of these proteins using ESEEM (electron spin echo envelope modulation) spectroscopy. The results show that neither of the three proteins influence the histidine ligation of manganese. From this, the conserved histidine of the 23 kDa protein can be ruled out as a manganese ligand. Whereas the 33 and 17 kDa proteins lack conserved histidines, the existence of a 33 kDa protein-derived carboxylate ligand has been posited; our results show no evidence for a change of the manganese co-ordination upon removal of this protein. Studies of the pH-dependence of the histidine–manganese coupling show that the histidine ligation is present in PS II centers showing the S₂ multiline EPR signal in the pH-range 4.2–9.5. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative studies with EPR and MRI on the in vivo tissue redox status estimation using redox-sensitive nitroxyl probes: influence of the choice of the region of interest

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T₁-weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T₁-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T₁-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300?MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T₁-weighted MRI and the T₁-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.     

Determination of the orientation of the axial ligands and of the magnetic properties of the haems in the tetrahaem ferricytochrome from Shewanella frigidimarina

The unambiguous assignment of the nuclear magnetic resonance (NMR) signals of the alpha-substituents of the haems in the tetrahaem cytochrome isolated from Shewanella frigidimarina NCIMB400, was made using a combination of homonuclear and heteronuclear experiments. The paramagnetic (13)C shifts of the nuclei directly bound to the porphyrin of each haem group were analysed in the framework of a model for the haem electronic structure. The analysis yields g-tensors for each haem, which allowed the assignment of some electron paramagnetic resonance (EPR) signals to specific haems, and the orientation of the magnetic axes relative to each haem to be established. The orientation of the axial ligands of the haems was determined semi-empirically from the NMR data, and the structural results were compared with those of the homologous tetrahaem cytochrome from Shewanella oneidensis MR-1 showing significant similarities between the two proteins.