Binding of ferric heme by the recombinant globin from the cyanobacterium Synechocystis sp. PCC 6803.

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin is a low-spin complex whose endogenous hexacoordination gives rise to optical and NMR characteristics reminiscent of cytochrome b(5) [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. In this work, the sequential assignments using (15)N-(13)C-labeled protein, (1)H nuclear Overhauser effects, and longitudinal relaxation data identified His70 as the proximal histidine and His46 as the sixth ligand to the iron ion. It was also found that one of two possible heme orientations within the protein matrix is highly preferred (>90%) and that this orientation is the same as in vertebrate myoglobins. The rate constant for the 180 degrees rotation of the heme within a protein cage to produce the favored isomer was 0.5 h(-1) at 25 degrees C, approximately 35 times faster than in sperm whale myoglobin. Variable temperature studies revealed an activation energy of 132 +/- 4 kJ mol(-1), similar to the value in metaquomyoglobin at the same pH. The rate constant for heme loss from the major isomer was estimated to be 0.01 h(-1) by optical spectroscopy, close to the value for myoglobin and decades slower than in the related Nostoc commune cyanoglobin. The slow heme loss was attributed in part to the additional coordination bond to His46, whereas the relatively fast rate of heme reorientation suggested that this bond was weaker than the proximal His70-Fe bond. The standard reduction potential of the hexacoordinated protein was measured with and without poly-L-lysine as a mediator and found to be approximately -150 mV vs SHE, indicating a stabilization of the ferric state compared to most hemoglobins and b(5) cytochromes.

Structural and dynamic perturbations induced by heme binding in cytochrome b5.

The water-soluble domain of rat hepatic cytochrome b(5) undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b(5) shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.

Salt effects on ionization equilibria of histidines in myoglobin.

The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.

Conformational properties of native sperm whale apomyoglobin in solution.

Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JalphaH-NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X-PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H-helix. The EF-F-FG segment remains uncharacterized.

Backbone dynamics of apocytochrome b5 in its native, partially folded state.

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.

The tautomeric state of histidines in myoglobin.

1H-15N HMQC spectra were collected on 15N-labeled sperm whale myoglobin (Mb) to determine the tautomeric state of its histidines in the neutral form. By analyzing metaquoMb and metcyanoMb data sets collected at various pH values, cross-peaks were assigned to the imidazole rings and their patterns interpreted. Of the nine histidines not interacting with the heme in sperm whale myoglobin, it was found that seven (His-12, His-48, His-81, His-82, His-113, His-116, and His-119) are predominantly in the N epsilon2H form with varying degrees of contribution from the Ndelta1 H form. The eighth, His-24, is in the Ndelta1H state as expected from the solid state structure. 13C correlation spectra were collected to probe the state of the ninth residue (His-36). Tentative interpretation of the data through comparison with horse Mb suggested that this ring is predominantly in the Ndelta1H state. In addition, signals were observed from the histidines associated with the heme (His-64, His-93, and His-97) in the 1H-15N HMQC spectra of the metcyano form. In several cases, the tautomeric state of the imidazole ring could not be derived from inspection of the solid state structure. It was noted that hydrogen bonding of the ring was not unambiguously reflected in the nitrogen chemical shift. With the experimentally determined tautomeric state composition in solution, it will be possible to broaden the scope of other studies focused on the electrostatic contribution of histidines to the thermodynamic properties of myoglobin.

Temperature dependence of histidine ionization constants in myoglobin.

The standard enthalpy of ionization of six titratable histidines in horse metaquomyoglobin was determined by repeating proton NMR titrations as a function of temperature and using the van't Hoff relationship. It was found that deltaH degrees varies between 16 and 37 kJ mol(-1) in the protein, compared with a value of 29 kJ mol(-1) in free histidine. The standard entropy change was evaluated by combining the enthalpy and free energy changes derived from the pKa values. Although the entropy change could not be precisely and accurately obtained by this method, it could be established that it spans a wide range, from -60 to 0 J K(-1) mol(-1), about the value of -23 J K(-1) mol(-1) for the free histidine. The entropy change was used within the framework of enthalpy-entropy compensation to partition the solvation component from the standard thermodynamic quantities for each of the titrating residues. It was shown that the partitioning of the values in the protein is not readily understood in terms of solvent accessibility or electrostatic interactions. The contribution of solvation effects to the temperature response appeared to be significant only in the case of His-119 and His-48. The standard quantities were also used to explore the energetics of proton binding in the native state at temperatures below the onset of thermal denaturation.

The native state of apomyoglobin described by proton NMR spectroscopy: the A-B-G-H interface of wild-type sperm whale apomyoglobin.

Proton nuclear magnetic resonance spectroscopy was applied to sperm whale apomyoglobin to describe the conformation adopted by the protein under native conditions. The study focused on the A-B-G-H interface, a region known to form a compact subdomain in the apoprotein (Hughson and Baldwin, Biochemistry 28:4415-4422, 1989). Two histidine residues located in this subdomain, His24 and His119, interact and are thought to play a role in the acid denaturation process (Barrick et al., J. Mol. Biol. 237:588-601, 1994). A stable double mutant at these positions (His24Val/His119Phe sperm whale apomyoglobin) was compared with wild-type apomyoglobin. The amino acid replacements result in chemical shift perturbations near the mutations, in particular in the AB interhelical region, and in a deceleration of backbone amide hydrogen exchange in the B helix from position 27 to position 33. The double mutant data were used to expand and confirm the wild-type spectral analysis. Signals from the D helix were identified that demonstrate the formation of holoprotein-like structure. The assigned wild-type nuclear Overhauser effects, although in small number, were sufficient to construct a model of the compact subdomain of the apoprotein. This was achieved by using the structure of the holoprotein and restraining it with the geometrical information on the apoprotein in a simulated annealing procedure. The experimental restraints define a low-resolution model of the A-B-G-H interface in apomyoglobin.

Design challenges for hemoproteins: the solution structure of apocytochrome b5.

In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.

1H and 15N NMR assignments of PsaE, a photosystem I subunit from the cyanobacterium Synechococcus sp. strain PCC 7002.

PsaE is a highly conserved, water-soluble protein of the photosystem I reaction center complexes of cyanobacteria, algae, and green plants. Along with the PsaC and PsaD proteins, the PsaE protein binds to the stromal surface of photosystem I and is required for cyclic electron transport in Synechococcus sp. strain PCC 7002 [Yu, L., Zhao, J., Mühlenhoff, U., Bryant, D.A., & Golbeck, J.H. (1993) Plant Physiol. 103, 171-180]. The psaE gene from this cyanobacterium encodes a mature protein of 69 amino acid residues and has recently been overexpressed in Escherichia coli [Zhao, J., Snyder, W.B., Mühlenhoff, U., Rhiel, E., Warren, P. V., Golbeck, J. H., & Bryant, D. A. (1993) Mol. Microbiol. 9, 183-194]. By using both unlabeled and uniformly 15N-labeled protein in a series of two- and three-dimensional NMR experiments, complete 1H and 15N amide resonance assignments were made. The major secondary structural element of PsaE is a five-stranded antiparallel beta-sheet. The five strands extend as follows: beta A, residues 7-10; beta B, residues 21-26; beta C, residues 36-39; beta D, residues 57-60; and beta E, residues 65-68. The topology is represented by (+1, +1, +1, -4x); it brings the first and last strands, and consequently the N- and C-termini, together. The protein has an extensive hydrophobic core organized around a conserved phenylalanine residue (Phe-40); another of its distinctive features is a segment extending from residue 42 to residue 56 devoid of dipolar contacts with the beta-sheet. The pK1/2 of the sole histidine residue (His-63) was determined to be 5.4.

Three-dimensional solution structure of PsaE from the cyanobacterium Synechococcus sp. strain PCC 7002, a photosystem I protein that shows structural homology with SH3 domains.

PsaE is a 69 amino acid polypeptide from photosystem I present on the stromal side of the thylakoid membrane. The three-dimensional solution structure of this protein from the cyanobacterium Synechococcus sp. strain PCC 7002 was determined at pH 5.8 and room temperature using over 900 experimental restraints derived from two- and three-dimensional NMR experiments. The structure is comprised of a well-defined five-stranded beta-sheet with (+1, +1, +1, -4 alpha) topology. There is no helical region except for a single turn of 3(10) helix between the beta D and beta E strands. PsaE also exhibits a large unrestrained loop spanning residues 42-56. A comparison to known protein structures revealed similarity with the Src homology 3 (SH3) domain, a membrane-associated protein involved in signal transduction in eukaryotes. The match is remarkable as 47 of the alpha-carbons of PsaE can be superimposed onto those of the SH3 domain from chicken brain alpha-spectrin with a root-mean-square deviation of 2.3 A. Although the amino acid sequences have low identity and the loops are different in both proteins, the topology of the beta-sheet and the 3(10) turn is conserved. SH3 domains from other sources show a similar structural homology. The structure of PsaE was used to suggest approaches for elucidating its roles within photosystem I.

The native state of apomyoglobin described by proton NMR spectroscopy: interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS.

Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His-64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His-82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481-6491). The packing of the side chains and the low pKa of His-64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter-amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.

Characterization of an independent structural unit in apocytochrome b5.

Apocytochrome b5 is a partially folded protein which contains a stable structural unit under native conditions [Moore, C.D., Al-Misky, O.N., & Lecomte, J.T.J. (1990) Biochemistry 30, 8357-8365]. In this work, the fold of the unit was examined by using 1H and 15N-edited two-dimensional NMR spectroscopy. It was found that it contains four of the five beta-strands and two of the six alpha-helices present in the holoprotein. The remainder of the structure appears to be mostly unstructured and fluctuating among several conformations. The structural unit is stabilized by a hydrophobic core formed by residues from each of the folded elements of secondary structure. Nuclear Overhauser effects and chemical shift values demonstrated that the unit is structurally similar in the apo- and holoproteins. However, the backbone amide hydrogen exchange was found to be much accelerated in the apoprotein. The paramagnetic relaxation agent HyTEMPO was used to probe the packing of the structure. HyTEMPO has unrestricted access to the empty heme binding site whereas it is unable to penetrate the stabilizing core. It was concluded that addition of the heme is necessary for the last strand to dock properly to the rest of the sheet. The kinetics of refolding of the apoprotein were monitored by stopped-flow fluorescence spectroscopy. Extensive protection of the sole tryptophan residue by docking of the two polypeptide termini occurs in less than 60 ms. It was proposed that apocytochrome b5, with its two-region behavior, might serve as a model for the design of proteins which bind a prosthetic group.

Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy.

Proton NMR spectroscopy was applied to myoglobin in the ferric, water-liganded form (metMbH2O) and the apo form (apoMb) to probe the structure and stability of the latter. Proteins from sperm whale and horse skeletal muscles were studied to simplify the spectral assignment task. Nuclear Overhauser effects and the response of chemical shifts to variations of pH were used as indicators of residual native holoprotein structure in the apoprotein. The investigation was focused in the histidine side chains and their environment. In metMbH2O, the resonances of all imidazole rings not interacting with the heme were assigned by applying standard two-dimensional methods. These assignments were found to differ from those reported elsewhere [Carver, J. A., & Bradbury, J. H. (1984) Biochemistry 23, 4890-4905] except for His-12, -113, and -116. Only one histidine (His-36) has a pK(a) higher than 7, two (His-48 and His-113) have a pK(a) lower than 5.5, and two (His-24 and His-82) appear not to titrate between pH 5.5 and pH 10. In the apoproteins, the signals of His-113 and His-116, as well as those of His-24, -36, -48, and -119 previously assigned in the horse globin [Cocco, M. J.. & Lecomte, J. T. J. (1990) Biochemistry 29, 11067-11072], could be followed between pH 5 and pH 10. A comparison to the holoprotein data indicated that heme removal has limited effect on the pK(a) and the surroundings of these residues. Five additional histidines which occur in the two helices and connecting loops forming the heme binding site were identified in the horse apoprotein. Four of these were found to have pK(a) values lower than that expected of an exposed residue. The NOE and titration data were proposed to reflect the fact that several holoprotein structural elements, in particular outside the heme binding site, are maintained in the apoprotein. In the heme binding region of the apoprotein structure, the low pK(a)'s suggest local environments which are resistant to protonation.

Similarities in structure between holocytochrome b5 and apocytochrome b5: NMR studies of the histidine residues.

The properties of the six histidine residues of apocytochrome b5 have been investigated by using one- and two-dimensional proton NMR spectroscopy in order to probe the structure remaining after heme removal. Spectral assignments were arrived at by analyzing proton NOE connectivities, comparing them to those observed in the holoprotein, and inspecting the X-ray structure of the latter species. Each histidine residue was studied for its pKa value, interaction with the relaxation agent copper nitrilotriacetic acid, and reactivity toward bromoacetic acid. The four histidines which are not coordinated to the iron atom in the holoprotein (His-15, -26, -27, and -80) display in the major conformer of the apoprotein the same characteristic properties as in the holoprotein. Three of them are involved in specific interactions with the rest of the structure: His-15 and His-80 participate in hydrogen bonds, and His-27 is influenced by the nearby C-terminal segment. His-26 is the most exposed to the solvent. His-63 and His-39, which are located in the heme binding site, have distinct pKa values; they are affected differently by the copper agent and exhibit comparable reactivity toward bromoacetic acid, albeit milder than that of His-26. The results show that the heme binding residues are clearly distinguishable by their physicochemical properties and that several elements of native holoprotein structure are in place in the apoprotein. It is proposed that the structural influence of the heme is localized and that the amino- and carboxy-terminal segments form a structural unit providing stability to the apoprotein and supporting a fluctuating, partially folded binding site.