Cytochrome oxidase from Pseudomonas aeruginosa. II. Reaction with copper protein

The reaction between a cytochrome oxidase and a copper protein from Pseudomonas aeruginosa has been studied by a rapid mixing technique. The data support the view that a complex is formed rapidly between the two proteins and is followed by a transfer of electrons in either direction. Reduced copper protein donates an electron to the heme c moiety of the oxidase with an apparent first-order rate constant of about 30 s⁻¹ while the transfer in the reverse direction proceeds with a constant of about 120 s⁻¹. The reaction between the copper protein and the heme c of the oxidase is followed by a much slower internal reaction involving electron transfer between the heme c and heme d.

The kinetic data have been analyzed in terms of the thermodynamics of the interactions. This analysis indicates that the copper protein has a ΔE of about 0.038 V more positive than the heme c component, a value that compares favorably with that of 0.040 V obtained by equilibrium methods. The value of ΔE obtained by the kinetic method for the internal reaction is less precise but is reasonably close to that of 0.070 V determined by an equilibrium technique.     

Isolation and partial characterization of the cytochrome c oxidase of Micrococcus luteus (lysodeikticus)

The cell membrane of Micrococcus luteus (lysodeikticus) contains a respiratory chain composed of hemes a, b, and c, which contain 171, 457, and 407 pmol/mg protein, respectively. Cytochrome c oxidase, the heme a containing component, has been purified after solubilization in Triton X-100, by gel filtration on Sepharose 4B-CL ammonium sulfate precipitation and ion-exchange and affinity chromatographies on a yeast cytochrome c-Sepharose 4B column. The purified complex, which contains three polypeptides of apparent Mr 47,000, 31,000, and 19,000, has CN-sensitive ferrocytochrome c oxidase activity (Ki = 0.35 microM) and a characteristic absorption spectrum with maxima in the oxidized form at 595 and 426 nm and in the reduced form at 601 and 444 nm. The purified enzyme contains 17.4 nmol/mg protein and its copper content is 23.2 nmol/mg protein. The enzyme was purified about 100-fold with respect to its content in crude membranes. The total heme a yield, also with respect to crude membranes content, was 6.8%.     

Effects of engineering uphill electron transfer into the methylamine dehydrogenase-amicyanin-cytochrome c-551i complex

Within the methylamine dehydrogenase-amicyanin-cytochrome c-551i complex, electrons are transferred from tryptophan tryptophylquinone (TTQ) to heme via the type I copper center of amicyanin. Mutation of Pro94 of amicyanin to Phe increases the redox potential of the copper center within the protein complex by approximately 195 mV. This introduces a large energy barrier for the second electron transfer (ET) step in this three-protein ET chain. As a consequence of this mutation, the ET rate from TTQ to copper exhibits about a 6-fold increase and the ET rate from copper to heme exhibits about a 100-fold decrease. These changes in ET rate are consistent with the predictions of Marcus theory. Temperature dependence studies of these reactions indicate that the reorganization energies for the ET to and from the copper center are unchanged by the P94F mutation, despite the large change in redox potential that it causes. Steady-state kinetic studies indicate that despite the large energy barrier for the ET from copper to heme, methylamine-dependent reduction of heme by the three-protein complex with P94F amicyanin goes to completion. The turnover number for this steady-state reaction, however, is decreased 50-fold relative to that of the native complex. As a consequence of the P94F mutation, the rate constant for the unfavorable uphill ET reaction from copper to heme has become the rate-limiting step in the overall reaction. The evolutionary implications of the effects of this mutation on the function of this naturally occurring simple ET chain are discussed.     

Molecular aspects of bovine erythrocyte bis[(heme b)copper] protein

An erythrocyte Cu2(heme b)2 protein of Mr 400000 was successfully isolated. Incubating the protein in sodium dodecyl sulfate prior to polyacrylamide gel electrophoresis caused the splitting into Mr 70000, 120000, and 200000 units. The copper was fully electron paramagnetic resonance detectable of the type II (g perpendicular = 2.0309, g parallel = 2.2122, A parallel = 175 G). The high-spin (d55/2) iron(III) showed a g value of 6.05. No magnetic interaction between copper and heme iron was detected. In a comparison of more than ten different enzymatic oxidase activities, not a single one could be assigned to the Cu2(heme b)2 protein. Azide, CO, cyanide, fluoride, and imidazole were bound to the heme iron. The binding of imidazolate suggests the accessibility of the sixth coordination site of the heme b group to fairly large ligands. Removal of the copper by ethylenediaminetetraacetic acid or cyanide resulted in an irreversible precipitation of the protein. This supports the structural contribution of the copper.     

Studies on the resolution of cytochrome oxidase

Cytochrome oxidase has been resolved in acetic acid and high salt/detergent media. In 0.5% acetic acid, the smaller subunits of the enzyme are selectively extracted with retention of an insoluble protein fraction containing subunits I–IV, VII. This fraction retains all the heme and copper of the original enzyme in a spectrally unaltered state, and possesses enzymic activity comparable to the unresolved enzyme. The further removal of subunit IV from this fraction results in migration of heme and copper and modification of their spectral characteristics. Resolution of the enzyme in a high salt/detergent medium extracts smaller subunits (V–VII) together with subunit IV and some heme and copper. The heme associated with this enzymically active extract has spectral characteristics that are partially suggestive of hemea ₃. It is suggested that the fraction of subunits I–IV, VII, resolved in dilute acetic acid, may represent the limit of resolution of the cytochrome oxidase complex that remains actively and spectrally indistinguishable from the original enzyme.     

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.     

Identification of the Mitochondrial Heme Metabolism Complex

Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.     

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.     

Components of cytochrome c oxidase detectable by EPR spectroscopy

A procedure for the preparation from frozen beef heart mitochondria of cytochrome c oxidase (EC 1.9.3.1) of high heme ( 14 μmoles/mg protein) and low extraneous copper ( 1.1 atoms Cu/mole heme) and low lipid ( 0.05 g phospholipid/g protein) content is described. EPR signals observed with the enzyme between 6 and 100 °K at various states of oxidation and at different conditions of pH and presence of solutes are described in detail. The quantities of paramagnetic species represented by these signals are estimated. Under no conditions does the sum of the EPR detectable species represent more than approx. 50% of the potentially paramagnetic components of the enzyme. Comparisons are made to the corresponding signals as observed in whole tissue, mitochondria and submitochondrial particles from a number of species. The assignment of the observed signals to known components of cytochrome c oxidase is discussed briefly.     

Metalloprotein complexes for the study of electron-transfer reactions. Characterization of diprotein complexes obtained by covalent cross-linking of cytochrome c and plastocyanin with a carbodiimide.

Cytochrome c (cyt) and zinc cytochrome c (Zncyt) are separately cross-linked to plastocyanin (pc) by the carbodiimide EDC according to a published method. The changes in the protein reduction potentials indicate the presence of approximately two amide cross-links. Chromatography of the diprotein complexes cyt/pc and Zncyt/pc on CM-52 resin yields multiple fractions, whose numbers depend on the eluent. UV-vis, EPR, CD, MCD, resonance Raman, and surface-enhanced resonance Raman spectra show that cross-linking does not significantly perturb the heme and blue copper active sites. Degrees of heme exposure show that plastocyanin covers most of the accessible heme edge in cytochrome c. Impossibility of cross-linking cytochrome c to a plastocyanin derivative whose acidic patch had been blocked by chemical modification shows that it is the acidic patch that abuts the heme edge in the covalent complex. The chromatographic fractions of the covalent diprotein complex are structurally similar to one another and to the electrostatic diprotein complex. Isoelectric points show that the fractions differ from one another in the number and distribution of N-acylurea groups, byproducts of the reaction with the carbodiimide. Cytochrome c and plastocyanin are also tethered to each other via lysine residues by N-hydroxysuccinimide diesters. Tethers, unlike direct amide bonds, allow mobility of the cross-linked molecules. Laser-flash-photolysis experiments show that, nonetheless, the intracomplex electron-transfer reaction cyt(II)/pc(II)----cyt(III)/pc(I) is undetectable in complexes of either type. Only the electrostatic diprotein complex, in which protein rearrangement from the docking configuration to the reactive configuration is unrestricted, undergoes this intracomplex reaction at a measurable rate.     

Decreased heme content and cessation of cell growth in cultured chick embryo fibroblasts in the presence of horse serum: Stimulation of heme synthesis and cell growth by iron

A new spectrofluorometric method for heme quantitation in cultured fibroblasts is described. The method includes: (1) heme extraction by methanol/sulfuric acid, (2) partial purification of heme by a microchromatographic method, and (3) treatment of the purified heme by oxalic acid followed by fluorometric quantitation. Using this method, heme concentration was determined in chick embryo fibroblasts cultured in a medium supplemented with either 7% fetal bovine serum (FBS) or 10% horse serum (HS). In the presence of FBS, cultured cells actively divided and cells contained 34–55 pmol heme/mg protein. In contrast, cultures maintained in HS proliferated at a slower rate and contained 23–25 pmol heme/mg protein. The addition of 40 μM FeSO₄ to cultures maintained in the presence of HS stimulated cell proliferation, and the cellular heme concentration increased to 37–51 pmol/ mg protein. These findings suggest that the cessation of growth in the presence of HS may be due to decreased heme content in the cells and that the stimulation of cell growth by iron is mediated by its stimulation of heme synthesis.     

革兰氏阴性菌血红素载体蛋白Hemophore的结构及作用机制

血红素作为宿主体内最丰富的铁离子来源,是致病菌营养竞争的主要目标,尤其对于血红素自身合成途径部分丧失的细菌。革兰氏阴性菌血红素转运系统由血红素载体蛋白(Hemophore)、外膜血红素受体、TonB-ExbB-ExbD复合物、ABC转运体等组成。Hemophore是存在于细菌细胞膜上或分泌到胞外环境中的一种蛋白。它能从宿主血红素结合蛋白中捕获血红素并将其传递给外膜受体。目前,在不同革兰氏阴性菌中已发现3种类型的Hemophore,分别是HasA、HxuA和HmuY型。本文将详细描述这3种Hemophore捕获血红素及与外膜受体相互作用的机制,以期为进一步研究其他细菌血红素载体蛋白的功能及作用机制奠定基础。     

血红素加氧酶系的共固定化及部分性质研究

为了检测血红素加氧酶系分子间的相互作用和共固定化酶系的反应动力学,利用２′,５′－ＡＤＰ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱对血红素加氧酶、ＮＡＤＰＨ－细胞色素ｃ还原酶和胆绿素还原酶进行非膜重组,再以纤维素为载体,用重氮化法固定此重组的酶复合物和共固定非重组的３种酶,发现固定化的重组酶系比非重组酶系能更好地发挥协同作用,在室温条件下可催化血红素一步合成胆红素．共固定化酶的最适ｐＨ为７．２,最适温度为３８℃,Ｋｍ值为０．９３μｍｏｌ／Ｌ．巯基试剂和金属卟淋对固定化酶有抑制作用,共固定化酶比游离酶系稳定性提高,３８℃下的操作半寿期可延长至４２０ｈ,在０～４℃保存两个月其酶活力无明显变化．     

The heme environment of mouse neuroglobin. Evidence for the presence of two conformations of the heme pocket

Neuroglobin (Ngb) is a newly discovered oxygen-binding heme protein that is primarily expressed in the brain of humans and other vertebrates. To characterize the structure/function relationships of this new heme protein, we have used resonance Raman spectroscopy to determine the structure of the heme environment in Ngb from mice. In the Fe(2+)CO complex, two conformations of the Fe-CO unit are present, one of which arises from an open conformation of the heme pocket in which the CO is not interacting with any nearby residue, and the other arises from a closed conformation where a positively charged residue near the CO group stabilizes the complex. For the Fe(2+)O(2) complex, we detect a single nu(Fe-OO) stretching mode at a frequency similar to that of oxymyoglobins and oxyhemoglobins of vertebrates (571 cm(-1)). Based on the Fe-C-O frequencies of the closed conformation of Ngb, a highly polar distal environment is indicated from which the O(2) off-rate is predicted to be lower than that of Mb. In the absence of exogenous ligands, a heme pocket residue coordinates to the heme iron, forming a six-coordinate complex, thereby predicting a low on-rate for exogenous ligands. These structural properties of the heme pocket of Ngb are discussed with respect to its proposed in vivo oxygen delivery function.     

The cytoplasmic heme-binding protein (PhuS) from the heme uptake system of Pseudomonas aeruginosa is an intracellular heme-trafficking protein to the delta-regioselective heme oxygenase

The uptake and utilization of heme as an iron source is a receptor-mediated process in bacterial pathogens and involves a number of proteins required for internalization and degradation of heme. In the following report we provide the first in-depth spectroscopic and functional characterization of a cytoplasmic heme-binding protein PhuS from the opportunistic pathogen Pseudomonas aeruginosa. Spectroscopic characterization of the heme-PhuS complex at neutral pH indicates that the heme is predominantly six-coordinate low spin. However, the resonance Raman spectra and global fit analysis of the UV-visible spectra show that at all pH values between 6 and 10 three distinct species are present to varying degrees. The distribution of the heme across multiple spin states and coordination number highlights the flexibility of the heme environment. We provide further evidence that the cytoplasmic heme-binding proteins, contrary to previous reports, are not heme oxygenases. The degradation of the heme-PhuS complex in the presence of a reducing agent is a result of H2O2 formed by direct reduction of molecular oxygen and does not yield biliverdin. In contrast, the heme-PhuS complex is an intracellular heme trafficking protein that specifically transfers heme to the previously characterized iron-regulated heme oxygenase pa-HO. Surface plasmon resonance experiments confirm that the transfer of heme is driven by a specific protein-protein interaction. This data taken together with the spectroscopic characterization is consistent with a protein that functions to shuttle heme within the cell.     

The mechanism of oxymyoglobin oxidation by copper ions and complexes: myoglobins carboxymethylated and carboxyamidated at histidine residues

The oxidation of sperm whale oxymyoglobin (MbO2) and its chemically modified derivatives alkylated at solvent-accessible histidines by sodium bromoacetate (CM-MbO2) and iodoacetamide (CA-MbO2) in the presence of ions and glycine complexes of copper, Cu2+, Cu(Gly)+, and Cu(Gly)2, has been studied. The influence of the reagent concentration, pH, and ionic strenth of medium, and also of competitive redox-inactive zinc ions on the reaction was investigated. The localization of Cu(Gly)2 in native sperm whale met-Mb and CM-met-Mb was examined by the high-resolution NMR method. The data obtained confirm that the linkage of copper compounds to surface histidines (all of them are away from the heme, at a distance of 1.8-2.7 nm) has only a minor (no more than 35%) contribution to the overall reaction rate, in particular under conditions of a large, more than 8-10-fold, data, excess of the reagent. The noticeable contribution of His116(113), His48, and His81, which according to NMR are localized on the protein surface and have the greatest affinity to copper, is revealed only at small concentrations of copper, a less than 5-fold excess relative to the protein. This is supported by the sigmoidal pH-dependence curve with the transition pK 6.5 at the equimolar copper concentration. The main contribution to the rate of the reaction studied should involve a linkage of copper to internal histidines, His97(FG4), which is 0.66 nm apart from the heme, and to distal His64(E7). Both are hydrogen bonded, the first with carboxyl group of one of the heme propionates, and the second with liganded O2, have a much lower affinity to copper than surface histidines, and are inaccessible to the modification.     

Biogenesis of respiratory cytochromes in bacteria.

Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible.     

Modeling electron transfer thermodynamics in protein complexes: interaction between two cytochromes c(3)

Redox protein complexes between type I and type II tetraheme cytochromes c(3) from Desulfovibrio vulgaris Hildenborough are here analyzed using theoretical methodologies. Various complexes were generated using rigid-body docking techniques, and the two lowest energy complexes (1 and 2) were relaxed using molecular dynamics simulations with explicit solvent and subjected to further characterization. Complex 1 corresponds to an interaction between hemes I from both cytochromes c(3). Complex 2 corresponds to an interaction between the heme IV from type I and the heme I from type II cytochrome c(3). Binding free energy calculations using molecular mechanics, Poisson-Boltzmann, and surface accessibility methods show that complex 2 is more stable than complex 1. Thermodynamic calculations on complex 2 show that complex formation induces changes in the reduction potential of both cytochromes c(3), but the changes are larger in the type I cytochrome c(3) (the largest one occurring on heme IV, of approximately 80 mV). These changes are sufficient to invert the global titration curves of both cytochromes, generating directionally in electron transfer from type I to type II cytochrome c(3), a phenomenon of obvious thermodynamic origin and consequences, but also with kinetic implications. The existence of processes like this occurring at complex formation may constitute a natural design of efficient redox chains.     

Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome<Emphasis Type="Italic"> c</Emphasis>551i as detected by EPR spectroscopy

EPR studies of the methylamine dehydrogenase (MADH)–amicyanin and MADH–amicyanin–cytochrome c551i crystalline complexes have been performed on randomly oriented microcrystals before and after exposure to the substrate, methylamine, as a function of pH. The results show that EPR signals from the redox centers present in the various proteins can be observed simultaneously. These results complement and extend earlier studies of the complexes under similar conditions that utilized single-crystal polarized absorption microspectrophotometry. The binary complex shows a blue copper axial signal, characteristic of oxidized amicyanin. After reaction of substrate with the MADH coenzyme tryptophan tryptophylquinone (TTQ), the binary complex exhibits an equilibrium mixture of oxidized copper/reduced TTQ and reduced copper/TTQ· radical, whose ratio is dependent on the pH. In the oxidized ternary complex, the same copper axial signal is observed superimposed on the low-spin ferric heme features characteristic of oxidized cytochrome c551i. After addition of substrate to the ternary complex, a decrease of the copper signal is observed, concomitant with the appearance of the radical signal derived from the semiquinone form of TTQ. The equilibrium distribution of electrons between TTQ and copper as a function of pH is similar to that observed for the binary complex. This result was essential to establish that the copper center retains its function within the crystalline ternary complex. At high pH, with time the low-spin heme EPR features disappear and the spectrum indicates that full reduction of the complex by substrate has occurred.