Showing papers in "Journal of Biological Inorganic Chemistry in 1997"

Myoglobin discriminates between O2, NO, and CO by electrostatic interactions with the bound ligand

Abstract: Most biological substrates have distinctive sizes, shapes, and charge distributions which can be recognized specifically by proteins. In contrast, myoglobin must discriminate between the diatomic gases O2, CO, and NO which are apolar and virtually the same size. Selectivity occurs at the level of the covalent Fe-ligand complexes, which exhibit markedly different bond strengths and electrostatic properties. By pulling a water molecule into the distal pocket, His64(E7)1 inhibits the binding of all three ligands by a factor of ∼10 compared to that observed for protoheme-imidazole complexes in organic solvents. In the case of O2 binding, this unfavorable effect is overcome by the formation of a strong hydrogen bond between His64(E7) and the highly polar FeO2 complex. This favorable electrostatic interaction stabilizes the bound O2 by a factor of ∼1000, and the net result is a 100-fold increase in overall affinity compared to model hemes or mutants with an apolar residue at position 64. Electrostatic interaction between FeCO and His64 is very weak, resulting in only a two- to three-fold stabilization of the bound state. In this case, the inhibitory effect of distal pocket water dominates, and a net fivefold reduction in K CO is observed for the wild-type protein compared to mutants with an apolar residue at position 64. Bound NO is stabilized ∼tenfold by hydrogen bonding to His64. This favorable interaction with FeNO exactly compensates for the tenfold inhibition due to the presence of distal pocket water, and the net result is little change in K NO when the distal histidine is replaced with apolar residues. Thus, it is the polarity of His64 which allows discrimination between the diatomic gases. Direct steric hindrance by this residue plays a minor role as judged by: (1) the independence of K O2, K CO, and K NO on the size of apolar residues inserted at position 64, and (2) the observation of small decreases, not increases, in CO affinity when the mobility of the His64 side chain is increased. Val68(E11) does appear to hinder selectively the binding of CO. However, the extent is no more than a factor of 2–5, and much smaller than electrostatic stabilization of bound O2 by the distal histidine.

Structural similarity and functional diversity in diiron-oxo proteins

Abstract: Diiron-oxo proteins currently represent one of the most rapidly developing areas of bioinorganic chemistry. All of these proteins contain a four-helix bundle protein fold surrounding a (μ-carboxylato)diiron core, and most, if not all, of the diiron(II) sites appear to react with O2 as part of their functional processes. Despite these common characteristics, an emerging functional diversity is one of the most striking aspects of this class of proteins. X-ray crystal structures of diiron(II) sites are now available for four of these proteins: hemerythrin (Hr), the hydroxylase protein of methane monooxygenase (MMOH), the R2 protein of Escherichia coli ribonucleotide reductase (RNR-R2), and a plant acyl-carrier protein Δ9-desaturase. The structure of the diiron(II) site in Hr, the sole O2 carrier in the group, is clearly distinct from the other three, whose function is oxygen activation. The Hr diiron site is more histidine rich, and the oxygen-activating diiron sites contain a pair of (D/E)X30–37EX2H ligand sequence motifs, which is clearly not found in Hr. The Hr diiron site apparently permits only terminal O2 coordination to a single iron, whereas the oxygen-activating diiron(II) centers present open or labile coordination sites on both irons of the center, and show a much greater coordinative flexibility upon oxidation to the diiron(III) state. Intermediates at the formal FeIIIFeIII and FeIVFeIV oxidation levels for MMOH and formal FeIIIFeIV oxidation level for RNR-R2 have been identified during reactions of the diiron(II) sites with O2. An [Fe2(μ-O)2]4+, 3+ "diamond core" structure has been proposed for the latter two oxidation levels. The intermediate at the FeIIIFeIV oxidation level in RNR-R2 is kinetically competent to generate a stable, functionally essential tyrosyl radical. The FeIVFeIV oxidation level is presumed to effect hydroxylation of hydrocarbons in MMOH, but the mechanism of this hydroxylation, particularly the involvement of discrete radicals, is currently controversial. The biological function of diiron sites in three members of this class, rubrerythrin, ferritin and bacterioferritin, remains enigmatic.

Molybdenum Active Centre of Dmso Reductase from Rhodobacter Capsulatus: Crystal Structure of the Oxidised Enzyme at 1.82-A Resolution and the Dithionite-Reduced Enzyme at 2.8-A Resolution

Abstract: The 1.82-A X-ray crystal structure of the oxidised (Mo(VI)) form of the enzyme dimethylsulfoxide reductase (DMSOR) isolated from Rhodobacter capsulatus is presented. The structure has been determined by building a partial model into a multiple isomorphous replacement map and fitting the crystal structure of DMSOR from Rhodobacter sphaeroides to the partial model. The enzyme structure has been refined, at 1.82-A resolution, to an R factor of 14.8% (R free = 18.4%). The molybdenum is coordinated by seven ligands: four dithiolene sulfurs, Oγ of Ser147 and two oxo groups. The four sulfur ligands, at a metal-sulfur distance of 2.4 A or 2.5 A, are contributed by the two molybdopterin guanine dinucleotide (MGD) cofactors. The coordination sphere of the molybdenum is different from that in previously reported structures of DMSOR from R. sphaeroides and R. capsulatus. The 2.8-A structure of DMSOR, reduced by addition of sodium dithionite, is also described and differs from the structure of the oxidised enzyme by the removal of a single oxo ligand from the molybdenum coordination sphere. A structure, at 2.5-A resolution, has also been obtained from crystals soaked in mother liquor buffered at pH 7.0. No differences are observed in the structure at pH 7 when compared with the native crystal structure at pH 5.5.

An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity

Abstract: The role of ceruloplasmin as a ferroxidase in the blood, mediating the release of iron from cells and its subsequent incorporation into serum transferrin, has long been the subject of speculation and debate. However, a recent X-ray crystal structure determination of human ceruloplasmin at a resolution of around 3.0 A, in conjunction with studies associating mutations in the ceruloplasmin gene with systemic haemosiderosis in humans, has added considerable weight to the argument in favour of a ferroxidase role for this enzyme. Further X-ray studies have now been undertaken involving the binding of the cations Co(II), Fe(II), Fe(III), and Cu(II) to ceruloplasmin. These results give insights into a mechanism for ferroxidase activity in ceruloplasmin. The residues and sites involved in ferroxidation are similar to those proposed for the heavy chains of human ferritin. The nature of the ferroxidase activity of human ceruloplasmin is described in terms of its three-dimensional molecular structure.

Desulfoferrodoxin Structure Determined by MAD Phasing and Refinement to 1.9 Angstroms Resolution Reveals a Unique Combination of a Tetrahedral Fes4 Centre with a Square Pyramidal Fesn4 Centre

Abstract: The structure of desulfoferrodoxin (DFX), a protein containing two mononuclear non-heme iron centres, has been solved by the MAD method using phases determined at 2.8 A resolution. The iron atoms in the native protein were used as the anomalous scatterers. The model was built from an electron density map obtained after density modification and refined against data collected at 1.9 A. Desulfoferrodoxin is a homodimer which can be described in terms of two domains, each with two crystallographically equivalent non-heme mononuclear iron centres. Domain I is similar to desulforedoxin with distorted rubredoxin-type centres, and domain II has iron centres with square pyramidal coordination to four nitrogens from histidines as the equatorial ligands and one sulfur from a cysteine as the axial ligand. Domain I in DFX shows a remarkable structural fit with the DX homodimer. Furthermore, three β-sheets extending from one monomer to another in DFX, two in domain I and one in domain II, strongly support the assumption of DFX as a functional dimer. A calcium ion, indispensable in the crystallisation process, was assumed at the dimer interface and appears to contribute to dimer stabilisation. The C-terminal domain in the monomer has a topology fold similar to that of fibronectin III.

Phenoxyl-copper(II) complexes: models for the active site of galactose oxidase

Abstract: The reaction of the macrocycles 1,4,7-tris (3,5-di-tert-butyl-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L1H3, or 1,4,7-tris(3-tert-butyl-5-methoxy-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L2H3, with Cu(ClO4)2·6H2O in methanol (in the presence of Et3N) affords the green complexes [CuII(L1H)] (1), [CuII(L2H)]·CH3OH (2) and (in the presence of HClO4) [CuII(L1H2)](ClO4) (3) and [CuII(L2H2)] (ClO4) (4). The CuII ions in these complexes are five-coordinate (square-base pyramidal), and each contains a dangling, uncoordinated pendent arm (phenol). Complexes 1 and 2 contain two equatorially coordinated phenolato ligands, whereas in 3 and 4 one of these is protonated, affording a coordinated phenol. Electrochemically, these complexes can be oxidized by one electron, generating the phenoxyl-copper(II) species [CuII(L1H)]+ ·, [Cu(L2H)]+ ·, [CuII(L1H2)]2+ ·, and [CuII(L2H2)]2+ ·, all of which are EPR-silent. These species are excellent models for the active form of the enzyme galactose oxidase (GO). Their spectroscopic features (UV-VIS, resonance Raman) are very similar to those reported for GO and unambiguously show that the complexes are phenoxyl-copper(II) rather than phenolato-copper(III) species.

Microscopic and semimacroscopic redox calculations: what can and cannot be learned from continuum models

Abstract: The major role of electrostatic effects in the control of redox potentials in proteins is now widely appreciated. However, the evaluation and conceptualization of the actual electrostatic contributions is far from trivial, and some models still overlook the nature of electrostatic effects in proteins. This commentary considers different contributions to redox potentials of proteins and discusses the ability of different models to capture these contributions. It is pointed out that macroscopic models which consider the protein as a medium of uniform low dielectric constant cannot reproduce the proper physics of redox proteins. In particular, it is pointed out that the crucial effects of the protein permanent dipoles must be taken into account explicitly and that these permanent dipoles involve effective dielectric constants that are different from those for ionized residues. It is also argued that the reorganization of the protein upon change of oxidation states or ionization of protein residues should be taken into account in redox calculations. The role of water penetration and the inadequacy of describing electrostatic effects by solvent accessibility is briefly mentioned. The nature and the meaning of the "dielectric constant" that should be used in redox calculations are also discussed. It is pointed out that the "dielectric constant" ep used in current discretized continuum (DC) models is simply a representation of the contributions which are treated implicitly and not the proper dielectric constant of the protein. It is then explained that the need to use a large "dielectric constant" in DC models reflects, among other factors, the implicit representation of the reorganization of permanent dipoles, and that even an explicit treatment of the fluctuations of ionized surface residues will lead to incorrect results when one uses ep=e¯ in continuum treatments. Finally, it is suggested that although the discussion and classification of different contributions to redox potentials is very useful, only the evaluation of the totality of the protein contributions (rather than an arbitrary subset) can lead to a quantitative understanding of redox proteins.

Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases

Abstract: Native nitrite reductases (NIRs) containing both type 1 and 2 Cu ions and type 2 Cu-depleted (T2D) NIRs from three denitrifying bacteria (Achromobacter cycloclastes IAM 1013, Alcaligenes xylosoxidans NCIB 11015, and Alcaligenes xylosoxidans GIFU 1051) have been characterized by electronic absorption, circular dichroism, and electron paramagnetic resonance spectra. The characteristic visible absorption spectra of these NIRs are due to the type 1 Cu centers, while the type 2 Cu centers hardly contribute in the same region. The intramolecular electron transfer (ET) process from the type 1 Cu to the type 2 Cu in native NIRs has been observed as the reoxidation of the type 1 Cu(I) center by pulse radiolysis, whereas no type 1 Cu in T2D NIRs exhibits the same reoxidation. The ET process obeys first-order kinetics, and observed rate constants are 1400–1900 s–1 (t1/2 = ca. 0.5 ms) at pH 7.0. In the presence of nitrite, the ET process also obeys first-order kinetics, with rate constants decreased by factors of 1/12–1/2 at the same pH. The redox potential of the type 2 Cu site is estimated to be +0.24 - +0.28 V, close to that of the type 1 Cu site. Nitrate and azide ions bound to the type 2 Cu site change the redox potential. Nitrite also would shift the redox potential of the type 2 Cu by coordination, and hence the intramolecular ET rate constant is decreased. Pulse radiolysis experiments on T2D NIRs in the presence of nitrite demonstrate that the type 1 Cu(I) site is slowly oxidized with a first-order rate constant of 0.03 s–1 at pH 7.0, suggesting that nitrite bound to the protein accepts an electron from the type 1 Cu. This result is in accord with the finding that T2D NIRs show enzymatic activities, although they are lower than those of the native enzymes.

Control of metalloprotein redox potentials: what does site-directed mutagenesis of hemoproteins tell us?

Abstract: An overview of factors affecting metalloprotein redox potentials is given, together with an assessment of the role of site-directed mutagenesis of hemoproteins in investigating this topic and a description of several theoretically challenging hemoproteins. The conclusion is reached that a quantitative and experimentally testable description of metalloprotein redox potentials is not yet available.

A first model for the oxidized active form of the active site in galactose oxidase: a free-radical copper complex

Abstract: Copper(II) complexes derived from the tripodal ligand bis(3′-t–butyl-2′-hydroxybenzyl)(2-pyridylmethyl)amine (LH2) have been studied in order to mimic the redox active site of the free radical-containing copper metalloenzyme galactose oxidase. In non-coordinating solvents such as dichloromethane, only an EPR-silent dimeric complex was obtained (L2Cu2). The crystal structure of L2Cu2 revealed a "butterfly" design of the [Cu(μOR)2Cu] unit, which is not flattened and leads to a short Cu–Cu distance, the t–butyl groups being localized on the same side of the [Cu(μOR)2Cu] unit. The dimeric structure was broken down by acetonitrile or by alcohols, leading quantitatively to a brown mononuclear copper(II) complex. UV-visible and EPR data indicated the coordination of the solvent in these mononuclear complexes. Electrochemical as well as chemical (silver acetate) one-electron oxidation of acetonitrile solutions of the monomeric complex led to a yellow-green solution. Based on EPR, UV-visible and resonance Raman spectroscopy, the one-electron oxidation product was identified as a cupric phenoxyl radical system. It slowly decomposes into a product where the ligand has been substituted (dimerization) in the para position of the hydroxyl group, for one of the phenolic groups. The data for the one-electron oxidized species provides strong evidence for a free-radical copper (II) complex.

Chromium in carbohydrate and lipid metabolism

Abstract: Since the discovery in the 1950s that mammals have a nutritional requirement for chromium, the biological function of chromium has been sought. Candidates for the naturally-occurring biologically active form of chromium have been proposed, but, until recently, all have been shown to be artifacts. Recent studies examining the properties of the oligopeptide low-molecular-weight chromium-binding substance (LMWCr) suggest that this material may have a role in carbohydrate and lipid metabolism as part of a novel insulin-signaling amplification mechanism and may have implications in the treatment of diabetes and related conditions.

The importance of the protein in controlling the electrochemistry of heme metalloproteins: methods of calculation and analysis

Abstract: The importance of electrostatic effects in determining the free energy of redox reactions in proteins such as cytochromes and iron-sulfur complexes is well established. Several theoretical techniques have been used to analyze how the protein and its environment combine to produce the observed electrochemical midpoints. The free energy of changing the cofactor charge is influenced by the distribution of charges and dipoles in the protein, solvent and ions surrounding the protein, and by the redistribution of these charges and dipoles coupled to the reaction. An outline of a consistent view for calculating these effects will be presented and compared with other theoretical models. Heme redox potentials in yeast cytochrome c and the cytochrome subunit of photosynthetic reaction centers will be calculated to show how these protein structures produce the observed electrochemistry.

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

Abstract: A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM–1 s–1 at 25 °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature 17O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25 °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA.

Metallocenter assembly in nickel-containing enzymes

Abstract: The five known nickel-dependent enzymes include urease, hydrogenase, carbon monoxide dehydrogenase (and CO dehydrogenase/acetyl-coenzyme A synthase), methyl-S–coenzyme M reductase, and one class of superoxide dismutase. Consistent with their disparate functions, these Ni enzymes have distinct metallocenter structures that vary in Ni coordination geometry, number and types of metals, and the presence of additional components. Sophisticated cellular Ni processing systems have been devised to allow for specific and functional incorporation of Ni into these proteins. This review highlights several themes that are common to the enzyme activation processes and summarizes current concepts related to the enzyme-specific Ni assembly pathways.

Formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774: isolation and spectroscopic characterization of the active sites (heme, iron-sulfur centers and molybdenum)

Abstract: An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO2, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150 kDa (three different subunits: 88, 29 and 16 kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S]2+/1+ centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g max=2.050, g med=1.947, g min=1.896) and an [Fe-S] center II (g max=2.071, g med=1.926, g min=1.865). Mossbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S]2+/1+ centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.

Structure of putrebactin, a new dihydroxamate siderophore produced by Shewanella putrefaciens

Abstract: Shewanella putrefaciens is a bacterium implicated in oil pipeline corrosion and fish spoilage, and is one of very few isolated microorganisms able to use iron(III) as an electron acceptor. S. putrefaciens strain 200 produced a novel cyclic dihydroxamate siderophore, putrebactin, during aerobic growth. Putrebactin was determined to be 1,11-dihydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, a cyclic dimer of succinyl-(N-hydroxyputrescine), by 1H and 13C NMR spectroscopy, fast atom bombardment and chemical ionization mass spectrometry, and X-ray crystallography. The protonation constants of putrebactin were determined to be 8.82 and 9.71. Potentiometric titration of the ferric complex revealed a sharp equivalence point at 3.0 equivalents of base per mole of Fe(III), consistent with loss of 3 protons per equivalent of bound ferric ion, while Job's method of continuous variation supported a shift from 1 : 1 to 3 : 2 complex stoichiometry as a function of pH. Putrebactin is similar in structure to two other siderophores, bisucaberin and alcaligin, produced by unrelated bacteria.

Co-evolution of cytochrome c6 and plastocyanin, mobile proteins transferring electrons from cytochrome b6f to photosystem I

Abstract: Cytochrome c6 and plastocyanin are soluble metalloproteins that act as mobile carriers transferring electrons between the two membrane-embedded photosynthetic complexes cytochrome b6 f and photosystem I (PSI). First, an account of recent data on structural and functional features of these two membrane complexes is presented. Afterwards, attention is focused on the mobile heme and copper proteins – and, in particular, on the structural factors that allow recognition and confer molecular specificity and control the rates of electron transfer from and to the membrane complexes. The interesting question of why plastocyanin has been chosen over the ancient heme protein is discussed to place emphasis on the evolutionary aspects. In fact, cytochrome c6 and plastocyanin are presented herein as an excellent case study of biological evolution, which is not only convergent (two different structures but the same physiological function), but also parallel (two proteins adapting themselves to vary accordingly to each other within the same organism).

The effect of redox state on the folding free energy of azurin

Abstract: The unfolding of oxidized and reduced azurin by guanidine hydrochloride has been monitored by circular dichroism. Dilution experiments showed the unfolding to be reversible, and the equilibrium data have been interpreted in terms of a two-state model. The protein is stabilized by the strong metal binding in the native state, so that the folding free energy is as high as –52.2 kJ mol–1 for the oxidized protein. The reduced protein is less stable, with a folding free energy of –40.0 kJ mol–1. A thermodynamic cycle shows, as a consequence, that unfolded azurin has a reduction potential 0.13 V above that of the folded protein. This is explained by the bipyramidal site in the native fold stabilizing Cu(II) by a rack mechanism, with the same geometry being maintained in the Cu(I) form. In the unfolded protein, on the other hand, the coordination geometries are expected to differ for the two oxidation states, Cu(I) being stabilized by the cysteine thiol group in a linear or trigonal symmetry, whereas Cu(II) prefers oxygen ligands in a tetragonal geometry.

X-ray structure of recombinant horse L-chain apoferritin at 2.0 angstrom resolution: Implications for stability and function.

Abstract: The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0 A resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.

Redox-Bohr effect in electron/proton energy transduction: cytochrome c 3 coupled to hydrogenase works as a 'proton thruster' in Desulfovibrio vulgaris

Abstract: A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c 3, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement.

Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

Abstract: The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N2S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential.

Biological electron tunneling through native protein media

Abstract: Nature has engineered a universe of redox proteins to efficiently control the oxidation and reduction of substrates and to convert redox energy into a delocalized transmembrane proton gradient power source. Some rapid physiologically relevant electron transfers are rate limited by electron tunneling. Distance appears to be the principle means naturally selected to control the speed of electron tunneling; free energy and reorganization energy can play important auxiliary roles. Thus, an electron from a biological redox center can tunnel in any direction and is likely to reduce the closest redox center with a favorable free energy. Although it is clearly possible to facilitate electron tunneling by designing covalent bridges in the regions between donors and acceptors, this does not seem to be a strategy that evolution has used. Evolutionary mutagenic adjustment of a bridge-like quality of the amino acid medium may be difficult in the face of heavy selection on the folding, stability and other properties of the protein medium. Repositioning cofactors by even a few angstroms has more profound effects on promoting and retarding rates, independent of the structure of the amino acid medium.

The coordination chemistry and function of the molybdenum centres of the oxomolybdoenzymes

Abstract: The nature of the catalytic centres of the oxomolybdoenzymes is considered with particular reference to the results of recent protein crystallographic studies. The different nature of these centres, with one or two molecules of a special pyranopterin (molybdopterin) ligating the metal through a dithiolene group, the presence or absence of a nucleotide appended to the phosphate of the molybdopterin AND the variation in the coordination chemistry at the metal render the term "THE molybdenum cofactor" meaningless and confusing. Rather, there is a series of such cofactors, related by the common denominators of a single molybdenum atom bound to the dithiolene group of the molybdopterin and, at some stage in the catalytic cycle, at least one terminal oxo group. This Mo(O)(molybdopterin) moiety is considered to be the metal-centred functional unit (McFU) of the oxomolybdoenzymes. Variations in the coordination chemistry and, therefore, the properties of the metal centre occur with the binding of other ligands, which can include: a terminal oxo or sulfido group, OH– and/or H2O group(s), a second pterin, and/or a serine, a cysteine or selenocysteine group from the polypeptide backbone of the protein. The role of molybdopterin is considered with particular reference to its potential involvement in the various redox processes necessary for the operation of the catalytic cycles of these enzymes; special attention is given to the possible cooperativity between formally metal-based and pterin-based redox processes.

X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Abstract: Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The oxidised (MoVI) protein has been investigated in solution at 77 K; the Mo K-edge position (20006.4 eV) is consistent with the presence of MoVI and, in agreement with the protein crystallographic results, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71 A), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32 A and two at 2.47 A, and two O atoms, one at 1.92 A (considered to be H-bonded to Trp 116) and one at 2.27 A (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (MoVI) DMSO reductase at 77 K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enzyme produced by the addition of dimethylsulfide (DMS) to the oxidised (MoVI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4 eV and 20004.5 eV, respectively; these values, together with the lack of a significant presence of MoV in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of MoIV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05 A and one at 2.51 A, and four S atoms at 2.36 A. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69 A, one at 1.91 A and one at 2.11 A, plus four S atoms, two at 2.28 A and two at 2.37 A. The EXAFS and the protein crystallographic results for the DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to MoIV with an Mo-O bond of length 1.92 A.

X-Ray Structure of the Cambialistic Superoxide Dismutase from Propionibacterium Shermanii Active with Fe or Mn

Abstract: The first structure of a cambialistic superoxide dismutase (SOD) from Propionibacterium shermanii exhibiting similar activity with iron and with manganese was solved at a resolution of 1.6 A and 1.9 A respectively. Surprisingly, no obvious differences between the two SODs were observable. The protein crystallises as a homo dimer in the asymmetric unit. Because of the crystallographic symmetry, it forms a tetramer. Structures of both the manganese and the ferric form were solved using molecular replacement techniques and multiple isomorphous replacement. The tertiary structure is similar to that of the other superoxide dismutases, the metal being fivefold coordinated by three histidines, one aspartate and one water molecule. The second shell of residues consists of hydrophobic amino acids, histidines and two water molecules, which are assumed to be involved in both the catalytic activity and structural stability of this superoxide dismutase. This shell may also be responsible for the cambialistic behaviour. This work shows that the reason for the metal specificity is not trivial, although minor alterations in the metal environment might be responsible for this behaviour.

The molybdenum-cofactor: a crystallographic perspective

Abstract: The molybdenum-cofactor (Mo-co) consists of a mononuclear molybdenum or tungsten ion coordinated by one or two molybdopterin ligands. Crystallographic analyses have demonstrated that the molybdopterin ligands are tricyclic and nonplanar, and that they coordinate the metal through their dithiolene sulfurs. Additional ligands to the metal may be provided by amino acid side chains (including serine, cysteine and selenocysteine), as well as one or more nonprotein O or S ligands, such as oxo, hydroxo, and sulfido. The molybdopterin ligand may participate in the various electron transfer reactions associated with the catalytic mechanism of these proteins, as suggested by both oxidation state-dependent changes in the metal coordination environment and the molybdopterin structure, and by the interaction of the molybdopterin with other redox groups within Mo-co-containing enzymes.

Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

Abstract: The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase.

Reaction profile of the last step in cytochrome P-450 catalysis revealed by studies of model complexes

Abstract: A series of oxoiron(IV) porphyrin cation radical complexes was investigated as compound I analogs of cytochrome P-450. Both the spectroscopic features and the reactivities of the complexes in oxygen atom transfer to olefins were examined as a function of only one variable, the axial ligand trans to the oxoiron(IV) bond. The results disclosed two important kinetic steps – electron transfer from olefin to oxoiron(IV) and intramolecular electron transfer from metal to porphyrin radical – which are affected differently by the axial ligands. The large kinetic barrier of the latter step in the reaction of olefins with the perchlorato-bound oxoiron(IV) porphyrin cation radical complex enabled the trapping of a reaction intermediate in which the metal, but not the porphyrin radical, is reduced. The first electron transfer step is probably followed by σ-bond formation, which readily accounts for formation of isomerized organic products at low temperatures. It is finally postulated that part of the enhanced oxygenation activities of cytochrome P-450 monooxygenases and chloroperoxidases is due to a lowering of the energy barrier for the second electron transfer step via participation of their redox-active cysteinate ligand.

The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation

Abstract: Ferritins uniquely direct the vectorial transfer of hydrated Fe(II)/Fe(III) ions to a condensed ferric phase in the central cavity of the soluble protein. Secondary, tertiary and quaternary structure are conserved in ferritin, but only five amino acid residues are conserved among all known ferritins. The sensitivity of ferroxidation rates to small differences in primary sequence between ferritin subunits that are cell-specifically expressed or to the conservative replacement of the conserved tyrosine 30 residue was demonstrated by examining recombinant (frog) H-type (red blood cell predominant) and M-type subunit (liver predominant) proteins which are both fast ferritins; the proteins form two differently colored Fe(III)-protein complexes absorbing at 550 nm or 650 nm, respectively. The complexes are convenient reporters of Fe(III)-protein interaction because they are transient in contrast to the Fe(III)-oxy complexes measured in the past at 310–420 nm, which are stable because of contributions from the mineral itself. The A650-nm species formed 18-fold faster in the M-subunit protein than did the 550-nm species in H-subunit ferritin, even though all the ferroxidase residues are the same; the Vmax was fivefold faster but the Hill coefficents were identical (1.6), suggesting similar mechanisms. In H-subunit ferritin, substitution of phenylalanine for conserved tyrosine 30 (located in the core of the subunit four-helix bundle) slowed ferroxidation tenfold, whereas changing surface tyrosine 25 or tyrosine 28 had no effect. The Fe(III)-tyrosinate was fortunately not changed by the mutation, based on the resonance Raman spectrum, and remained a suitable reporter for Fe(III)-protein interactions. Thus, the A550/650 nm can also report on post-oxidation events such as transport through the protein. The impact of Y30F on rates of formation of Fe(III)-protein complexes in ferritin, combined with Mossbauer spectroscopic studies that showed the parallel formation of multiple Fe(III) postoxidation species (three dinuclear oxy and one trinuclear oxy species) (A. S. Periera et al., Biochemistry 36 : 7917–7927, 1997) and the loss of several of the multimeric Fe(III) post-oxidation species in a Y30F alteration of human recombinant H-ferritin (E. R. Bauminger et al., Biochem J. 296 : 709–719, 1993), indicate that at least one of the pathways for Fe oxidation/transfer in ferritin is through the center of the four-helix bundle and is influenced by structural features dependent on tyrosine 30.

Enzymes of heme biosynthesis

Abstract: Heme is a necessary component in a variety of oxygen-binding proteins and electron-transfer proteins, and as such it occupies a central role in cellular and organismal metabolism. With only rare exceptions, organisms that utilize heme possess the entire biosynthetic pathway to produce this tetrapyrrole compound. The enzymes involved catalyze a variety of interesting reactions and utilize both common and unique cofactors and metals. Aminolevulinate dehydratase from all organisms and ferrochelatase from higher animals are both metalloenzymes, while 5-aminolevulinate synthase contains pyridoxal phosphate, and porphobilinogen deaminase possesses a unique dipyrrole cofactor. Two pathway enzymes catalyze multiple decarboxylations and yet have no cofactors, and one enzyme catalyzes a six-electron oxidation with a single FAD. To add additional scientific interest there exist biochemically and clinically distinct human genetic diseases for every step in this pathway.