Nuclear magnetic spin-lattice relaxation of water protons caused by metal cage compounds.

The nuclear magnetic spin-lattice relaxation rates of water protons are reported for solutions of manganese(II), copper(II), and chromium(III) cage complexes of the sarcophagine type. As simple aqueous solutions, the complexes are only modest magnetic relaxation agents, presumably because they lack protons on atoms in the first-coordination-sphere protons that are sufficiently labile to mix the large relaxation rate at the metal complex with that of the bulk solvent. The relaxation is approximately modeled using spectral density functions derived for translational diffusion of the interacting dipole moments with the modification that the electron spin relaxation rate is directly included as a contribution to the correlation time. In all cases studied, the electron spin relaxation rate is sufficiently large that it contributes directly to the water-proton spin relaxation process. The poor relaxation efficiency of the cage compound may, however, be improved dramatically by binding the complex to a protein. The efficiency is improved even further if the rotational motion of the protein is reduced drastically by an intermolecular cross-linking reaction. The relaxation efficiency of the cross-linked protein-cage complexes rivals that of the best first-coordination-sphere relaxation agents like [Gd(DTPA)(H2O)]2- and [Gd(DOTA)(H2O)]-.     

Hemin: a possible cause of oxidative stress in blood circulation of beta-thalassemia/hemoglobin E disease

A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of beta-thalassemia/hemoglobin E disease (beta-thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 microM in serum of beta-thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of beta-thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of beta-thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum heroin, r = 0.778 (p < 0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p = 0.259) and 0.519 (p = 0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of beta-thal/Hb E.     

Autoxidation of the site-specifically PEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the autoxidation

The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.     

Cyanide binding to hexacoordinate cyanobacterial hemoglobins: hydrogen-bonding network and heme pocket rearrangement in ferric H117A Synechocystis hemoglobin

The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of (1)H(2)O/(2)H(2)O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the (15)N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed to the reactive 117 site of Synechocystis Hb as a potential determinant of biophysical and, perhaps, functional properties.     

Effect of Hb-encapsulation with vesicles on H2O2 reaction and lipid peroxidation

We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H(2)O(2)) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H(2)O(2), metHb (Fe(III)) and ferrylHb (Fe(IV)=O) are produced, and H(2)O(2) is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H(2)O(2) that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H(2)O(2). Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H(2)O(2), the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H(2)O(2), whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.     

Monitoring the reaction of hemoglobin with hydrogen peroxide by capillary electrophoresis-chemiluminescence detection

The reaction of hemoglobin (Hb) with hydrogen peroxide (H2O2) leads to fluorescent product and heme degradation. We applied capillary electrophoresis-chemiluminescence (CE-CL) detection to monitor the course of Hb reacting with H2O2. Hb and released free iron ion (Fe3+) were detected based on their enhancement effects on CL of the luminol-H2O2 system. In this study, we discovered an intermediate of this reaction which intensely enhances the luminol-H2O2 CL system. The ratio of max CL signals of Fe3+, Hb and this intermediate is circa 1:10:60.     

Proton nuclear Overhauser effect investigation of the heme pockets in ligated hemoglobin: conformational differences between oxy and carbonmonoxy forms

Proton nuclear Overhauser effect (NOE) measurements have been used extensively to investigate the detailed conformations of peptides, proteins, and nucleic acids in the solution state. However, much of the published work has dealth with molecules of molecular weight less than 15 000. It is generally thought that specific NOEs cannot be observed in larger molecules (due to spin diffusion), so that NOE is of little use in conformational studies of such systems. By use of truncated-driven NOE with an irradiation time of 100 ms, specific NOEs are observed in a protein of the size of human normal adult hemoglobin (Hb A, 65 000 daltons). This technique has permitted us to assign several proton proton resonances arising from heme groups and from amino acid residues situated in the vicinity of the ligand binding site (such as E7 histidine and E11 valine) of the alpha and beta chains of Hb A. In addition, two-dimensional 1H[1H] J-correlated spectroscopy (COSY) experiments as well as theoretical ring-current calculations have confirmed the spectral assignments obtained by the one-dimensional NOE experiments. These new results not only have permitted us to map the heme pockets and to investigate the conformational differences in the heme pockets between oxy and carbonmonoxy forms of Hb A but also have demonstrated that the technique of truncated-driven NOE can be used to investigate the detailed conformations of selected regions in larger macromolecules in a way heretofore thought not to be feasible.     

Monooxygenase activity of human hemoglobin: NMR demonstration of different modes of substrate binding corresponding to different activities of hemoglobin derivatives

In the accompanying paper [Ferraiolo, B. L., Onady, G. M., & Mieyal, J. J. (1984) Biochemistry (preceding paper in this issue)] we reported different aniline hydroxylase activities for ferrihemoglobin, its isolated subunits, and the converse pair of valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 in a reconstituted system containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) and cytochrome P-450 reductase. To investigate the molecular basis for the different activities, 1H NMR T1 relaxation studies of aniline were performed in the absence and presence of each of the hemoglobin (Hb) species. The paramagnetic contribution of the ferric heme iron atoms of each Hb derivative to the enhanced relaxation of the proton nuclei of aniline was determined relative to control experiments in which the hemoproteins had been converted fully to the corresponding (carbonmonoxy)ferrous forms, which are diamagnetic. According to the known distance dependence of the paramagnetic effect and the relative changes in T1 for the upfield and downfield signals in the spectrum of aniline, it was ascertained that aniline binds in the same manner to the beta-ferric hybrid and to ferrihemoglobin. These two forms displayed equivalent hydroxylase activities that were the highest among the Hb derivatives for the same aniline concentration. The T1 changes observed with the alpha-ferric hybrid suggest a different orientation for aniline in that complex. The T1 data for the isolated subunits alpha 3+ and beta 3+4 would indicate that overall binding of aniline includes a component of direct aniline-heme ligation in each case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine

The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction.     

MgFe-layered double hydroxide modified electrodes for direct electron transfer of heme proteins

In this study, the Fe-based layered double hydroxides (Mg(3)Fe LDH) were used to immobilize heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) for fabrication of heme/Mg(3)Fe LDH film on glassy carbon electrode (Mg(3)Fe-heme/GCE). The possible role of iron in framework of LDH to promote direct electron transfer (DET) of heme proteins was investigated using an LDH containing non-iron as a reference. Hb was selected as a model protein for studying the electrocatalytic activity of immobilized heme in LDH film. The Mg(3)Fe-Hb/GCE displayed an enhanced electrocatalytic reduction towards H(2)O(2). The biosensor showed a very low detection limit (0.036μM) and apparent Michaelis-Menten constant (7.98μM). This work outlines that Fe-based LDH modified electrode provides a promising platform for immobilization of heme proteins and development of sensitive biosensors.     

The heme complex of Hmu O, a bacterial heme degradation enzyme from Corynebacterium diphtheriae. Structure of the catalytic site.

Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK(a) of 9. (1)H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O(2) and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O(2). Mammalian heme oxygenase has only one O(2) orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart.     

Modulation of oxygen binding to insect hemoglobins: the structure of hemoglobin from the botfly Gasterophilus intestinalis

Hemoglobins (Hbs) reversibly bind gaseous diatomic ligands (e.g., O2) as the sixth heme axial ligand of the penta-coordinate deoxygenated form. Selected members of the Hb superfamily, however, display a functionally relevant hexa-coordinate heme Fe atom in their deoxygenated state. Endogenous heme hexa-coordination is generally provided in these Hbs by the E7 residue (often His), which thus modulates accessibility to the heme distal pocket and reactivity of the heme toward exogenous ligands. Such a pivotal role of the E7 residue is prominently shown by analysis of the functional and structural properties of insect Hbs. Here, we report the 2.6 A crystal structure of oxygenated Gasterophilus intestinalis Hb1, a Hb known to display a penta-coordinate heme in the deoxygenated form. The structure is analyzed in comparison with those of Drosophila melanogaster Hb, exhibiting a hexa-coordinate heme in its deoxygenated derivative, and of Chironomus thummi thummi HbIII, which displays a penta-coordinate heme in the deoxygenated form. Despite evident structural differences in the heme distal pockets, the distinct molecular mechanisms regulating O2 binding to the three insect Hbs result in similar O(2 affinities (P50 values ranging between 0.12 torr and 0.46 torr).     

Structural basis of peroxide-mediated changes in human hemoglobin: a novel oxidative pathway

Hydrogen peroxide (H(2)O(2)) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H(2)O(2) to highly purified human hemoglobin (HbA(0)) induced structural changes that primarily resided within beta subunits followed by the internalization of the heme moiety within alpha subunits. These modifications were observed when an equal molar concentration of H(2)O(2) was added to HbA(0) yet became more abundant with greater concentrations of H(2)O(2). Mass spectrometric and amino acid analysis revealed for the first time that betaCys-93 and betaCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA(0) was treated with H(2)O(2). Oxidation of further amino acids in HbA(0) exclusive to the beta-globin chain included modification of betaTrp-15 to oxyindolyl and kynureninyl products as well as betaMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the beta subunits as a result of the H(2)O(2) attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two alpha-peptide fragments (alpha128-alpha139) and a heme moiety with the loss of iron, cross-linked between alphaSer-138 and the porphyrin ring. The novel oxidative pathway of HbA(0) modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.     

Monoamine-dependent production of reactive oxygen species catalyzed by pseudoperoxidase activity of human hemoglobin

Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, beta-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O2*-). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc'). The O2*- generation and Asc formation were detected by O2*--specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O2*- production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H2O2, but a trace of H2O2 added to the system greatly increased the production of both O2*- and Asc*. Addition of GSH completely inhibited the PEA-dependent production of O2*- and Asc* in Hb solution. We propose that the O2*- generation and Asc* formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.     

Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin

Neuroglobin (Ngb) and cytoglobin (Cygb) are two recently discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve-globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb is held to facilitate O2 diffusion to the mitochondria and to protect neuronal cells from hypoxic-ischemic insults, may be an oxidative stress-responsive sensor protein for signal transduction, and may carry out enzymatic activities, such as NO/O2 scavenging. Cygb is linked to collagen synthesis, may provide O2 for enzymatic reactions, and may be involved in a ROS(NO)-signaling pathway(s). Ngb and Cgb display the classical three-over-three alpha-helical fold of Hb and Mb, and are endowed with a hexa-coordinate heme-Fe atom, in their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous ligand. Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of Ngb and Cygb. Moreover, Ngb and Cygb display a tunnel/cavity system within the protein matrix held to facilitate ligand channeling to/from the heme, multiple ligand copies storage, multi-ligand reactions, and conformational transitions supporting ligand binding.     

Structural characterization of nitrimyoglobin

Nitrimyoglobin was formed in greater than 94% yield by a simple reaction between excess nitrite and horse heart metmyoglobin at pH 5.5. This dark green pigment was shown by 1H NMR spectroscopy to be a single, pure product with a well defined tertiary structure that is highly similar to the starting myoglobin. Electronic spin states parallel those of myoglobin, although the relaxation times differ. Ligand binding reactions of nitrimyoglobin parallel those of normal myoglobin, but lead to a unique series of UV-visible spectra. In the ferrous state, nitrimyoglobin reversibly binds O2 with half-saturation of sites at an O2 partial pressure of 10.4 +/- 1.4 mm Hg. 1H NMR data indicate that the altered heme of nitrimyoglobin has not undergone reaction at any meso proton position, nor has it been partially saturated to the level of a chlorin. 15N NMR spectra indicate that only a single nitrogen was added to the protein as a nitro group. Extraction of the modified heme from nitrimyoglobin and spectroscopic characterization of the nitriheme by infrared spectroscopy and of the free base porphyrin methyl ester derived from nitriheme by 1H NMR indicate that the modification is regiospecific. The heme in nitrimyoglobin is 3-(trans-2-nitrovinyl)-2,7,12,18-tetramethyl-8-vinylporphyrin-13,1 7-dipropionic acid. In the Fisher nomenclature scheme, the 2-vinyl substituent is the site of modification and has been converted to a nitrovinyl group by substitution of a proton by -NO2.     

NMR relaxation of protein and water protons in diamagnetic hemoglobin solutions

We have measured T1 and T2 of protein and water protons in hemoglobin solutions using broad-line pulse techniques; selective excitation and detection methods enabled the intrinsic protein and water relaxation rates, as well as the spin-transfer rate between them, to be obtained at 5, 10, and 20 MHz. Water and protein T1 data were also obtained at 100 and 200 MHz for hemoglobin in H2O/D2O mixtures by using commercial Fourier-transform instruments. The T1 data conform to a simple model of two well-mixed spin systems with single intrinsic relaxation times and an average spin-transfer rate, with each phase recovering from a radio-frequency excitation with a biexponential time dependence. At low frequencies, protein T1 and T2 agree reasonably with a model of dipolar relaxation of an array of fixed protons tumbling in solution, explicitly calculating methyl and methylene relaxation and using a continuum approximation for the others. Differing values in H2O and D2O are mainly ascribed to solvent viscosity. For water-proton relaxation, T1, T2, and spin transfer were measured for H2O and HDO, which enabled a separation of inter-and intramolecular contributions to relaxation. Despite such detail, few firm conclusions could be reached about hydration water. But it seems clear that few long-lived hydration sites are needed to explain T1 and T2, and the spin-transfer value mandates fewer than five sites with a lifetime longer than 10(-8) s.     

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.     

Assignment of 1H and 13C hyperfine-shifted resonances for tuna ferricytochrome c.

Tuna ferricytochrome c has been used to demonstrate the potential for completely assigning 1H and 13C strongly hyperfine-shifted resonances in metalloprotein paramagnetic centers. This was done by implementation of standard two-dimensional NMR experiments adapted to take advantage of the enhanced relaxation rates of strongly hyperfine-shifted nuclei. The results show that complete proton assignments of the heme and axial ligands can be achieved, and that assignments of several strongly shifted protons from amino acids located close to the heme can also be made. Virtually all proton-bearing heme 13C resonances have been located, and additional 13C resonances from heme vicinity amino acids are also identified. These results represent an improvement over previous proton resonance assignment efforts that were predicated on the knowledge of specific assignments in the diamagnetic protein and relied on magnetization transfer experiments in heterogeneous solutions composed of mixtures of diamagnetic ferrocytochrome c and paramagnetic ferricytochrome c. Even with that more complicated procedure, complete heme proton assignments for ferricytochrome c have never been demonstrated by a single laboratory. The results presented here were achieved using a more generally applicable strategy with a solution of the uniformly oxidized protein, thereby eliminating the requirement of fast electron self-exchange, which is a condition that is frequently not met.