Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase.

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

Elevated nitric oxide/peroxynitrite mechanism for the common etiology of multiple chemical sensitivity, chronic fatigue syndrome, and posttraumatic stress disorder.

Various types of evidence implicate nitric oxide and an oxidant, possibly peroxynitrite, in MCS and chemical intolerance (CI). The positive feedback loops proposed earlier for CFS may explain the chronic nature of MCS (CI) as well as several of its other reported properties. These observations raise the possibility that this proposed elevated nitric oxide/peroxynitrite mechanism may be the mechanism of a new disease paradigm, answering the question raised by Miller earlier: "Are we on the threshold of a new theory of disease?"

Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes.

A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants.

Temperature, pH, and solvent isotope effects on cytochrome c peroxidase mutant N82A studied by proton NMR.

The mutant of baker's yeast cytochrome c peroxidase-CN with Ala82 in place of Asn82, [N82A]CcPCN, exhibits a complex solution behavior featuring dynamic interconversion among three enzyme forms that so far have only been detected by NMR spectroscopy. Proton NMR studies of [N82A]CcPCN reveal resonances from each of the three enzyme forms and show that the interconversion among forms is controlled by the pH, temperature, and isotope composition (H2O vs. D2O) of the buffer solution. No evidence for a key hydrogen bond between His52 and heme-coordinated cyanide is found in any of the enzyme forms, indicating that disruption of the extensive distal hydrogen bonding network is the source of this phenomenon.

Solution structure and backbone dynamics of component IV Glycera dibranchiata monomeric hemoglobin-CO.

The solution structure and backbone dynamics of the recombinant, ferrous CO-ligated form of component IV monomeric hemoglobin from Glycera dibranchiata (GMH4CO) have been characterized by NMR spectroscopy. Distance geometry and simulated annealing calculations utilizing a total of 2550 distance and torsion angle constraints yielded an ensemble of 29 structures with an overall average backbone rmsd of 0.48 A from the average structure. Differences between the solution structure and a related crystal structure are confined to regions of lower precision in either the NMR or X-ray structure, or in regions where the amino acid sequences differ. 15N relaxation measurements at 76.0 and 60.8 MHz were analyzed with an extended model-free approach, and revealed low-frequency motions in the vicinity of the heme, concentrated in the F helix. Amide proton protection factors were obtained from H-D amide exchange measurements on 15N-labeled protein. Patterns in the backbone dynamics and protection factors were shown to correlate with regions of heterogeneity and disorder in the ensemble of NMR structures and with large crystallographic B-factors in the X-ray structures. Surprisingly, while the backbone atoms of the F helix have higher rmsds and larger measures of dynamics on the microsecond to millisecond time scale than the other helices, amide protection factors for residues in the F helix were observed to be similar to those of the other helices. This contrasts with H-D amide exchange measurements on sperm whale myoglobin which indicated low protection for the F helix (S. N. Loh and B. F. Volkman, unpublished results). These results for GMH4 suggest a model in which the F helix undergoes collective motions as a relatively rigid hydrogen-bonded unit, possibly pivoting about a central position near residue Val87.

Detailed NMR analysis of the heme-protein interactions in component IV Glycera dibranchiata monomeric hemoglobin-CO.

Complete 13C, 15N, and 1H resonance assignments have been obtained for the recombinant, ferrous CO-ligated from of component IV monomeric hemoglobin from Glycera dibranchiata. This 15642 Da myoglobin-like protein contains a large number of glycine and alanine residues (47) and a heme prosthetic group. Coupling constant information has allowed the determination of chi(1) and chi(2) torsion angles, backbone phi angles, as well as 43 of 81 possible assignments to H beta 2/beta 3 pairs. The 13C alpha, 13 beta, 13C', and 1H alpha assignments yield a consensus chemical shift index (CSI) that, in combination with NOE information and backbone torsion angles, defines seven distinct helical regions for the protein's global architecture. Discrepancies between the CSI and NOE/3JHNH alpha-based secondary structure definitions have been attributed to heme ring current shifts on the basis of calculations from a model structure [Alam et al. (1994) J. Protein Chem., 13, 151-164]. The agreement can be improved by correcting the 1H alpha chemical shifts for the ring current contributions. Because the holoprotein was assembled from isotopically enriched globin and natural isotope-abundance heme, data from 13C-filtered/13C-edited and 13C-filtered/13C-filtered 2D NOESY experiments could be used to determine complete heme proton assignments and to position the heme within the protein. The results confirm the unusual presence of Phe31 (B10) and Leu58 (E7) side chains near the heme ligand binding site which may alter the polarity and steric environment and thus the functional properties of this protein.

Electrospray ionization mass spectrometric study of the multiple intracellular monomeric and polymeric hemoglobins of Glycera dibranchiata.

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500-15,200 Da) and an approximately equal number of polymer Hb peaks (15,500-16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; -2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2-4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.

Sequential main-chain proton and carbon resonance assignments for wild-type yeast iso-1 ferrocytochrome c and identification of secondary structural elements.

Yeast iso-1 cytochrome c is a naturally occurring protein that possesses an unusually reactive Cys102 that imbues iso-1 with a complicated solution chemistry which includes spontaneous dimerization and poorly characterized redox reactions. For this reason previous studies of this typical member of the c-type cytochromes have been relegated to variant proteins in which the 102 position has been mutated, with most common changes involving serine and threonine. However, we have determined sequential 1H resonance assignments for the wild-type native protein because it is the actual participant in yeast mitochondrial electron transfer processes and because the wild-type native protein should be the fundamental assignment basis. In addition to 1H resonance assignments for 97 of 106 amino acids, we have also provided an extensive database of long-range NOEs. Comparison of these NOEs and a chemical shift index based upon alpha-H resonances has lead to identification of solution secondary structural elements that are consistent with the solid-state crystal structure. Although there is currently no efficient expression system that would facilitate isotope labeling of iso-1 cytochrome c, we tried to assess the usefulness of future heteronuclear experiments by using natural-abundance 1H/13C HMQC experiments to unambiguously assign 35 alpha-C resonances.

Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-ferricytochrome c free in solution and bound to cytochrome c peroxidase.

Extensive proton hyperfine-shifted resonance assignments have been made for wild-type yeast iso-1-ferricytochrome c when it is free in solution and when it is noncovalently complexed to resting state cytochrome c peroxidase. Complete heme proton resonance assignments were made for free iso-1-ferricytochrome c, while for CcP-complexed iso-1-ferricytochrome c, 70% of heme proton assignments were made. Additional proton resonance assignments were made for hyperfine-shifted protons of amino acids near the heme. These assignments allowed identification of the most extensive set of complex-induced proton shifts yet reported for CcP/cytochrome c complexes. Several purely dipolar-shifted resonances from heme vicinity amino acid protons were also assigned in both free and complexed iso-1-ferricyt c. Both sets of resonance assignments allowed assessment of the origin of proton complex-induced shifts. Using the assigned dipolar-shifted proton resonances as a basis, the orientations of the principal axis systems of the paramagnetic susceptibility tensors for free and cytochrome c peroxidase-bound iso-1-ferricytochrome c were elucidated. The results indicated that the iso-1-ferricytochrome c magnetic axis system orientation shifts significantly upon complex formation. The direction of the complex-induced shifts for heme proton resonances is largely accounted for by the magnetic anisotropy changes. However, analysis of heme complex-induced shifts also reveals local changes in magnetic environment for two heme substituents, presumably through a specific structure change.

Structural features of Glycera dibranchiata monomer hemoglobins. Primary sequences of monomer hemoglobin components II and III.

Primary sequences for the remaining two members (GMH2, GMH3) of the group of three major monomeric hemoglobins from the marine annelid Glycera dibranchiata have been obtained. Full sequences of each 147-amino acid globin were achieved with a high degree of confidence using standard Edman technology in combination with molecular mass determinations of the intact globins and of the cyanogen bromide cleavage fragments using electrospray ionization mass spectrometry. When minor assumptions concerning Q/E identities are made these new results indicate the likely correspondence of GMG2 with the protein represented by the first Glycera dibranchiata monomer hemoglobin complete sequence [Imamura et al., (1972), J. Biol. Chem. 247, 2785-2797]. When these new sequences are combined with the previously determined primary sequence for the third major monomer hemoglobin, GMH4 [Alam et al., J. Protein Chem. (1994), 13, 151-164], it becomes clear that these three (GMG2-4) are truly distinct proteins, contrary to previous suggestions. Surprisingly, our results show that none of these three primary sequences is identical to the published sequence of the refined monomer hemoglobin crystal structure protein; however, there is a strong correspondence to the GMG2 sequence. The present sequencing results, in combination with the published GMH4 sequence, confirm the presence of a distal Leu in place of the more commonly encountered distal His in all three of the major monomer hemoglobins isolated in this laboratory and indicate that the unusual B10 Phe occurs only in GMH4. Analysis of the sequences presented here, along with comparison of amino acid content for Glycera dibranchiata monomer hemoglobins isolated from three different laboratories, and comparison of NMR results from two laboratories suggest further correspondence which unify disparate published isolations.

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.

Cytochrome c peroxidase complexed with cytochrome c has an unperturbed heme moiety.

Transient resonance Raman, Raman difference, circular dichroism (CD), and optical absorption studies have been carried out on the electrostatic complexes formed by yeast cytochrome c peroxidase (CCP) with horse cytochrome c (Cytc) in low ionic strength solutions. In all the complexes examined [e.g., CCP(II)/Cytc(II), CCP(III)/Cytc(II), CCP(III)/Cytc(III)], the local heme environments of both proteins are largely unperturbed upon complexation. Specifically, CCP preserves a completely pentacoordinate high-spin heme in both its ferric and ferrous forms in CCP/Cytc complexes and uncomplexed mixtures. We found no evidence corroborating the previously reported increase in the low-spin fraction of CCP heme upon complexation with Cytc [Hildebrandt et al. (1992) Biochemistry 31, 2384-2392]. Instead, our Raman data strongly suggest that the H-bonding networks in the distal and proximal pockets of CCP are well maintained in the complexes. On the other hand, CD spectra of CCP(III)/Cytc(III) complexes showed substantial variations (relative to the uncomplexed mixtures) in the far-UV region, reflecting some protein conformational rearrangements. In addition, the spectral data suggest that complexation with Cytc affects the previously observed pH-dependent flexibility of the heme structure of CCP and thus influences the photodynamics of the CCP active site.

Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants.

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.

A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein.

Derivatives of yeast iso-1 cytochrome c, chemically modified at Cys-102 (Cys-102 acetamide-derivatized monomer, Cys-102 thionitrobenzoate-derivatized monomer, Cys-102 S-methylated monomer, and the disulfide dimer), exhibit different spectral and physicochemical properties relative to the native, unmodified protein, depending on the nature of the modifying group. The results of proton NMR studies on the Cys-102 acetamide-derivatized monomer of iso-1 ferricytochrome c indicate that the conformational characteristics of the heme environment in this protein derivative are intermediate between those of the unmodified monomer and disulfide dimer forms of the protein. Measurements of the pKa of the alkaline transitions of the five forms of iso-1 ferricytochrome c provided values of 8.89, 8.82, 8.67, 8.47, and 8.50 for the unmodified monomer, S-methylated monomer, acetamide-derivatized monomer, thionitrobenzoate-derivatized monomer, and disulfide dimer, respectively. The results of proton NMR studies of the reduced form of these proteins suggest that the heme environments of the unmodified monomer and disulfide dimer derivatives of iso-1 ferrocytochrome c are similar and indicate that treatment of the thionitrobenzoate-derivatized and disulfide dimer forms of the protein with sodium dithionite results in cleavage of the disulfide bonds at position 102. Circular dichroism studies reveal that only the disulfide dimer form of iso-1 ferricytochrome c exhibits a Soret CD spectrum which differs from the native, unmodified monomer in that the intensity of the negative band at approximately 420 nm is diminished in the spectrum of the dimer relative to the spectrum of the monomer. Soret CD spectra of the ascorbate-reduced form of all protein derivatives are similar. The process of "autoreduction" of yeast iso-1 ferricytochrome c is shown to occur in the absence of a free sulfhydryl group at position 102 and is exacerbated under moderately high pH conditions. These results are suggestive of the presence of a redox-active amino acid, perhaps a tyrosine, in yeast iso-1 cytochrome c.

A peroxidative model of human erythrocyte intracellular Ca2+ changes with in vivo cell aging: measurement by 19F-NMR spectroscopy.

Numerous changes occur with human erythrocyte aging in vivo, including an increase in free ionic intracellular calcium concentration ([Ca2+]i) (N.R. Aiken et al. (1992) Biochim. Biophys. Acta 1136, 155-160). An attractive hypothesis of cell aging suggests that oxidative stress is responsible for many age-related changes. To determine whether oxidative stress leads to increased intracellular Ca2+ concentrations, we used the fluorinated calcium probe 5,5'-difluoroBAPTA and fluorine nuclear magnetic resonance spectroscopy (19F-NMR) to measure [Ca2+]i following mild hydrogen peroxide (H2O2) stress to young red cells. Cells were separated using density centrifugation, exposed to 815 microM H2O2, loaded with the calcium probe, and [Ca2+]i measured. Intracellular [Ca2+] increased from 62 nM (+/- 4, S.E.) in untreated young cells to 173 nM (+/- 11) in peroxide treated cohort young cells. This value approached our previously reported [Ca2+]i of 221 nM (+/- 25) in old human erythrocytes. Pretreatment of young cells with (a) cobalt, which blocks Ca2+ influx through calcium channels, or (b) carbon monoxide, which prevents methemoglobin formation, inhibited the peroxide-induced increase in ionic intracellular calcium. These findings are consistent with the hypothesis that oxidative stress of erythrocytes contributes to the increased [Ca2+]i found in senescent cells, and that this is due to increased membrane Ca2+ leak resulting from oxidatively induced methemoglobin-cytoskeletal protein crosslinking.

Studies of protein-protein association between yeast cytochrome c peroxidase and yeast iso-1 ferricytochrome c by hydrogen-deuterium exchange labeling and proton NMR spectroscopy.

Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.

The effect of the Asn82-->Asp mutation in yeast cytochrome c peroxidase studied by proton NMR spectroscopy.

Proton NMR studies of the mutant of baker's yeast cytochrome c peroxidase-cyanide with the Asn 82-->Asp mutation ([N82D]cytochrome c peroxidase-CN) are presented and compared to the wild-type enzyme. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is an important participant in the initial hydrogen peroxide decomposition step of cytochrome c peroxidase. In wild-type cytochrome c peroxidase-CN, His52 hydrogen bonds to the neighboring Asn82 peptide carbonyl group and to heme-coordinated cyanide. His52 thus manifests itself as an extensively hydrogen bonded histidinium moiety. The principal result from this study is the observation that three hyperfine-shifted resonances disappear from the spectrum of [N82D] cytochrome c peroxidase-CN compared to the wild-type enzyme. All three absent resonances in [N82D]cytochrome c peroxidase-CN belong to His52 and this leads to the conclusion that the result of the mutation has been elimination of the His52-Asn82 and His52-heme-coordinated cyanide hydrogen bonds.

Expression of recombinant monomer hemoglobins (component IV) from the marine annelid Glycera dibranchiata: evidence for primary sequence positional regulation of heme rotational disorder.

A description of the efficient high-level expression of the monomer hemoglobin (GMG4) from Glycera dibranchiata is presented. The cDNA described by Simons and Satterlee [Simons, P.C., & Satterlee, J.D. (1989) Biochemistry 28, 8525-8530] was subcloned into an expression system, and conditions were found that led to the production of large amounts of soluble apoprotein (rec-gmg). These conditions included lowering the temperature during the induction period and growth in a rich medium with a higher ionic strength. Characterization of this reconstituted recombinant protein showed that it was not identical to the native GMH4 protein. Both UV-visible and 1H NMR data indicated differences within the holoprotein (rec-gmh) heme pocket compared to the native protein, the major difference being that two nonidentical heme orientations are significantly populated in rec-gmh. This phenomenon has been seen previously in other heme proteins, where these heme orientational isomers are described by a 180-deg rotation about the heme alpha-gamma meso axis. This work prompted the production of a complete chemical sequence for the native GMH4 [Alam S.L., Satterlee, J. D., & Edmonds, C. G. (1994) J. Protein Chem. 13, 151-164], which showed that the expressed rec-gmg protein differed at three primary sequence positions (41, 95, and 123) from the native component IV globin (GMG4). Subsequently, we have produced the triple-revertant mutations required to express the recombinant wild-type protein (recGMG4). The physical characteristics of the active site in the holoprotein (recGMH4) are identical to those of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete heme proton hyperfine resonance assignments of the Glycera dibranchiata component IV metcyano monomer hemoglobin.

Monomer hemoglobin component IV is one of three major myoglobin-like proteins found in the erythrocytes of the marine annelid Glycera dibranchiata. Unlike myoglobin, all three of these monomer hemoglobin components lack the distal histidine, which is replaced by leucine. This substitution alters the protein's functional properties due to its proximity to the heme ligand binding site. As the initial step toward a full NMR characterization of this protein, a complete set of self-consistent proton NMR assignments for the heme and the proximal histidine of the paramagnetic, metcyano form of native component IV (metGMH4CN) is presented. These assignments relied upon a combination of one- and two-dimensional NMR spectroscopy, including nonselective spin-lattice relaxation time measurements. The metcyano form has been chosen for several reasons: (1) The heme paramagnetism acts as an intrinsic shift reagent which aids in making individual resonance assignments for the heme and neighboring amino acids in the protein's ligand binding site. (2) Heme paramagnetism also enhances proton nuclear relaxation rates, thereby allowing two-dimensional NMR experiments to be carried out at very rapid repetition rates (i.e., 5 s-1). (3) The heme proton hyperfine resonance pattern for this paramagnetic form of wild-type monomer hemoglobin component IV provides an analytical reference for the integrity of the heme active site. This is anticipated to facilitate rapid analysis of subsequently produced recombinant derivatives of this protein. (4) The cyanide-ligated protein has a heme pocket structure similar to those of the O2- and CO-ligated forms of the physiologically important, reduced form of the protein, so that the heme and proximal histidine proton assignments will serve as a basis for further assignments within the heme binding site. Complete assignments, in combination with recombinant derivatives of this monomer hemoglobin, will give further insight into local interactions that influence ligand binding kinetics and heme orientational isomerism.

Complete amino acid sequence of the Glycera dibranchiata monomer hemoglobin component IV: structural implications.

The globin derived from the monomer Component IV hemoglobin of the marine amnelid, Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reported Glycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structural model for monomer Component IV hemoglobin was constructed using the published 1.5 A crystal structure of a monomer hemoglobin from Glycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.