1H NMR investigation of the distal hydrogen bonding network and ligand tilt in the cyanomet complex of oxygen-avid Ascaris suum hemoglobin.

The O(2)-avid hemoglobin from the parasitic nematode Ascaris suum exhibits one of the slowest known O(2) off rates. Solution (1)H NMR has been used to investigate the electronic and molecular structural properties of the active site for the cyano-met derivative of the recombinant first domain of this protein. Assignment of the heme, axial His, and majority of the residues in contact with the heme reveals a molecular structure that is the same as reported in the A. suum HbO(2) crystal structure (Yang, J., Kloek, A., Goldberg, D. E., and Mathews, F. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4224-4228) with the exception that the heme in solution is rotated by 180 degrees about the alpha,gamma-meso axis relative to that in the crystal. The observed dipolar shifts, together with the crystal coordinates of HbO(2), provide the orientation of the magnetic axes in the molecular framework. The major magnetic axis, which correlates with the Fe-CN vector, is found oriented approximately 30 degrees away from the heme normal and indicates significant steric tilt because of interaction with Tyr(30)(B10). The three side chain labile protons for the distal residues Tyr(30)(B10) and Gln(64)(E7) were identified, and their relaxation, dipolar shifts, and nuclear Overhauser effects to adjacent residues used to place them in the distal pocket. It is shown that these two distal residues exhibit the same orientations ideal for H bonding to the ligand and to each other, as found in the A. suum HbO(2) crystal. It is concluded that the ligated cyanide participates in the same distal H bonding network as ligated O(2). The combination of the strong steric tilt of the bound cyanide and slow ring reorientation of the Tyr(30)(B10) side chain supports a crowded and constrained distal pocket.     

Solution 1H NMR study of the active site structure for the double mutant H64Q/V68F cyanide complex from mouse neuroglobin

Solution (1)H NMR spectroscopy has been carried out to investigate the molecular and electronic structures of the active site in H64Q/V68F double mutant mouse neuroglobin in the cyanomet form. Two heme orientations resulting from a 180 degrees rotation about the alpha-gamma-meso axis were observed with a population ratio about 1:1, and the clearly distinguished B isomer was used to perform the study. Based on the analysis of the dipolar shifts and paramagnetic relaxation constants, the distal Gln(64)(E7) side chain is obtained to adopt an orientation that may produce hydrogen bond between the N(epsilon)H(1) and the Fe-bound cyanide. The side chain of Phe(68)(E11) is oriented out of the heme pocket just like that in triple mutant of cyanide complex of sperm whale myoglobin. A 15 degrees rotation of the imidazole ring in axial His(96) is observed, which is close to the varphi angle determined from the crystal structure of NgbCO. The quantitative determinations of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that cyanide is tilted by 8 degrees from the heme normal which allows for contact to the Gln(64)(E7) N(epsilon)H(1). The E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand. Furthermore, the tilt of the ligand has no obvious influence on the heme heterogeneity of cyanide ligation for isomer A/B of the wild type and mutant protein, indicating that factors other than steric effects, such as polarity of heme pocket, impacts on ligand binding affinity.     

Solution (1)H NMR study of the influence of distal hydrogen bonding and N terminus acetylation on the active site electronic and molecular structure of Aplysia limacina cyanomet myoglobin

The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution (1)H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small approximately 2 degrees rotation of the axial His in rWT relative to true WT and a approximately 3 degrees rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix.     

Application of the paramagnetic dipole field for solution NMR active site structure determination in low-spin, cyanide-inhibited ferric hemoproteins

The principles for the application of the paramagnetic dipolar field of low-spin, cyanide-inhibited ferrihemoproteins for determining active site structure are briefly described. The ubiquitous dipolar shifts for assigned residues, together with crystal coordinates of some appropriate structural homolog, allow determination of the orientation and anisotropies of the paramagnetic dipolar tensor. The orientation of chi uniquely defines the orientation of the Fe-CN unit, which is tilted variably and sensitively monitors distal steric and H-bond interactions. The mapped dipolar field, in turn, can be used to determine the orientation of mutated residues. Case studies involving unusual genetic variants and point mutants of myoglobins, human hemoglobins, horseradish peroxidase and its substrate complex of heme oxygenase are presented as examples.     

Solution H NMR characterization of substrate-free C. diphtheriae heme oxygenase: Pertinence for determining magnetic axes in paramagnetic substrate complexes

Proton 2D NMR was used to confirm in solution a highly conserved portion of the molecular structure upon substrate loss for the heme oxygenase from the pathogenic bacterium Corynebacterium diphtheriae, HmuO. The chemical shifts for the conserved portion of the structure are assessed as references for the dipolar shifts needed to determine the orientation of the paramagnetic susceptibility tensor, χ, in paramagnetic substrate complexes of HmuO. It is shown that the chemical shifts for the structurally conserved portion of substrate-free HmuO serve as excellent references for residues with only small to moderate sized dipolar shifts in the cyanide-inhibited substrate complex of HmuO, yielding an orientation of χ that is essentially the same as conventionally obtained from large dipolar shifts based on empirical estimates of the diamagnetic reference. The implications of these diamagnetic chemical shifts for characterizing the hydrogen bonding in the physiologically relevant, resting-state, high-spin aquo complex are discussed. The pattern of labile proton exchange in the distal H-bond network of substrate-free HmuO allowed comparison of changes in dynamic stability of tertiary contacts in the substrate-free and substrate-bound HmuO and with the same complexes of human heme oxygenase.     

1H NMR investigation of the solution structure of substrate-free human heme oxygenase: comparison to the cyanide-inhibited, substrate-bound complex

1H NMR was used to investigate the molecular structure, and dynamic properties of soluble, recombinant, substrate-free human heme oxygenase (apohHO) on a comparative basis with similar studies on the substrate complex. Limited but crucial sequence-specific assignments identify five conserved secondary structural elements, and the detection of highly characteristic dipolar or H-bond interactions among these elements together with insignificant chemical shift differences confirm a strongly conserved folding topology of helices C-H relative to that of substrate complexes in either solution or the crystal. The correction of the chemical shifts for paramagnetic and porphyrin ring current influences in the paramagnetic substrate complex reveals that the strength of all but one of the numerous relatively robust H-bonds are conserved in apohHO, and similar ordered water molecules are located near these H-bond donors as observed in the substrate complexes. The unique and significant weakening of the Tyr(58) OH hydrogen bond to the catalytically critical Asp(140) carboxylate in apohHO is suggested to arise from the removal of the axial H-bond acceptor ligand rather than the loss of substrate. The interhelical positions of the conserved strong H-bonds argue for a structural role in maintaining a conserved structure for helices C-H upon loss of substrate. While the structure and H-bond network are largely conserved upon loss of substrate, the variably increased rate of NH lability dictates a significant loss of dynamic stability in the conserved structure, particularly near the distal helix F.     

1H NMR characterization of the solution active site structure of substrate-bound, cyanide-inhibited heme oxygenase from Neisseria meningitidis: comparison to crystal structures

Heme oxygenase, HO, from the pathogenic bacterium Neisseria meningitidis catabolizes heme for the iron necessary for infection. The enzyme, labeled HemO, exhibits less sequence homology to mammalian HO than another studied HO from Corynebacterium diphtheriae. Solution 1H NMR has been utilized to define the active site molecular and electronic structure of the cyanide-inhibited, substrate-bound complex for comparison with those provided by several crystal structures. Extensive assignments by solely 1H NMR 2D methods reveal a structure that is very strongly conserved with respect to the crystal structure, although 1H/2H exchange indicates dynamically much more stable distal and proximal helices than those for other HOs. Several residues found with alternate orientations in crystal structures of water- and NO-ligated complexes were shown to occupy positions found solely in the NO complex, confirming that there are structural accommodations in response to ligating the substrate complex with a diatomic, H-bond acceptor ligand. The observed dipolar shifts allow the determination of the magnetic axes that show that the Fe-CN unit is tilted approximately 10 degrees toward the alpha-meso position, thereby facilitating the alpha-stereoselectivity of the enzyme. Numerous labile protons with larger than usual low-field bias are identified and, in common with the other HO complexes, shown to participate in an extended, distal side H-bond network. This H-bond network orders several water molecules, most, but not all, of which have been detected crystallographically. A series of three C-terminal residues, His207-Arg208-His209, are not detected in crystal structures. However, 1H NMR finds two residues, His207 and likely Arg208 in contact with pyrrole D, which in crystal structures is exposed to solvent. The nature of the NOEs leads us to propose a H-bond between the proximally oriented His207 ring and the carboxylate of Asp27 and a salt-bridge between the terminus of Arg208 and the reoriented 7-propionyl carboxylate. While numerous ordered water molecules are found near both propionates in the crystal structure, we find much larger water NOEs to the 6- than 7-propionate, suggesting that water molecules near the 7-propionate have been expelled from the cavity by the insertion of Arg208 into the distal pocket. The conversion of the 7-propionate link from the N-terminal region (Lys16) to the C-terminal region (Arg208) in the ligated substrate complex both closes the heme cavity more tightly and may facilitate product exit, the rate-limiting step in the enzyme activity.     

1H NMR study of the effect of variable ligand on heme oxygenase electronic and molecular structure

Heme oxygenase carries out stereospecific catabolism of protohemin to yield iron, CO and biliverdin. Instability of the physiological oxy complex has necessitated the use of model ligands, of which cyanide and azide are amenable to solution NMR characterization. Since cyanide and azide are contrasting models for bound oxygen, it is of interest to characterize differences in their molecular and/or electronic structures. We report on detailed 2D NMR comparison of the azide and cyanide substrate complexes of heme oxygenase from Neisseria meningitidis, which reveals significant and widespread differences in chemical shifts between the two complexes. To differentiate molecular from electronic structural changes between the two complexes, the anisotropy and orientation of the paramagnetic susceptibility tensor were determined for the azide complex for comparison with those for the cyanide complex. Comparison of the predicted and observed dipolar shifts reveals that shift differences are strongly dominated by differences in electronic structure and do not provide any evidence for detectable differences in molecular structure or hydrogen bonding except in the immediate vicinity of the distal ligand. The readily cleaved C-terminus interacts with the active site and saturation-transfer allows difficult heme assignments in the high-spin aquo complex.     

NMR determination of the orientation of the magnetic susceptibility tensor in cyanometmyoglobin: a new probe of steric tilt of bound ligand

The experimentally determined paramagnetic dipolar shifts for noncoordinated amino acid side-chain protons in the heme pocket of sperm whale cyanometmyoglobin [Emerson, S. d., & La Mar, G. N. (1990) Biochemistry (preceding paper in this issue]) were used to determine in solution the orientation of the principal axes for the paramagnetic susceptibility tensor relative to the heme iron molecular coordinates. The determination was made by a least-squares search for the unique Euler rotation angles which convert the geometric factors in the molecular (crystal) coordinates to ones that correctly predict each of 41 known dipolar shifts by using the magnetic anisotropies computed previously [Horrocks, W. D., Jr., & Greenberg, E. S. (973) Biochim. Biophys. Acta 322, 38-44]. An excellent fit to experimental shifts was obtained, which also provided predictions that allowed subsequent new assignments to be made. The magnetic axes are oriented so that the z axis is tipped approximately 15 degrees from the heme normal toward the hem delta-meso-H and coincides approximately with the characterized FeCO tilt axis in the isostructural MbCO complex [Kuriyan, J., Wilz, S., Karplus, M., & Petsko, G. A. (1986) J. Mol. Biol. 192, 133-154]. Since the FeCO and FeCN units are isostructural, we propose that the dominant protein constraints that tips the magnetic z axis from the heme normal is the tilt of the FeCN by steric interactions with the distal residues. The rhombic magnetic axes were found to align closely with the projection of the proximal His imidazole plane on the heme, confirming that the His-Fe bonding provides the protein constraints that orients the in-plane anisotrophy. The tipped magnetic z axis is shown to account quantitatively for the previously noted major discrepancy between the hyperfine shift patterns for the bound imidazole side chain in models and protein. Moreover, it is shown that the proximal His ring nolabile proton hyperfine shifts provide direct and exquisitely sensitive indicators of the degree of the z axis tilt that may serve as a valuable probe for characterizing variable steric interactions in the distal pocket of both point mutants and natural genetic variants of myoglobin.     

One- and two-dimensional nuclear Overhauser effect studies of the electronic/molecular structure of the heme cavity of ferricytochrome b5

A nuclear Overhauser effect, NOE, study of solubilized native bovine ferricytochrome b5 has provided the complete assignment of the heme resonances as well as those of the majority of the amino acid side-chains making contact with the prosthetic group. The resonances which could not be identified are those from positions very close to the iron (less than 5 A) for which paramagnetic relaxation is sufficiently strong to significantly decrease the NOEs. The observed 1H-1H dipolar contacts generally confirm a solution structure unchanged from that described in single crystals, except for the detailed orientation of the heme side-chains. The 2-vinyl group is found in both the cis and trans in-plane orientation as opposed to exclusively cis in the crystal, and the 7-propionate group is rotated by 30 degrees in solution towards the 6-propionate group. Identification of resonances for the individual axial histidine residues indicates non-equivalent interaction with the heme iron, and the patterns of meso-H, pyrrole substituent and amino acid dipolar shifts allow the location of the principal magnetic axes in the protein coordinate system. This identifies His-39 as the dominant influence in determining the electronic ground state that orients the molecular orbital for facile electron transfer via the exposed heme edge. The complete two-dimensional NOESY map for ferricytochrome b5 is presented that yields all the cross peaks expected on the basis of the one-dimensional NOE studies, and indicates that such two-dimensional methods should have profitable extension to strongly hyperfine-shifted resonances in paramagnetic proteins.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A

The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional 1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the alpha-chain with its subunit in HbA. The target residues are those that exhibit significant (>0.2 ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than 0.2 ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in HbACO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet alpha-chain and that this approach should be similarly effective in HbA.     

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.     

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.     

NO binding induced conformational changes in a truncated hemoglobin from Mycobacterium tuberculosis

The resonance Raman spectra of the NO-bound ferric derivatives of wild-type HbN and the B10 Tyr --> Phe mutant of HbN, a hemoglobin from Mycobacterium tuberculosis, were examined with both Soret and UV excitation. The Fe-N-O stretching and bending modes of the NO derivative of the wild-type protein were tentatively assigned at 591 and 579 cm(-1), respectively. Upon B10 mutation, the Fe-NO stretching mode was slightly enhanced and the bending mode diminished in amplitude. In addition, the N-O stretching mode shifted from 1914 to 1908 cm(-1). These data suggest that the B10 Tyr forms an H-bond(s) with the heme-bound NO and causes it to bend in the wild-type protein. To further investigate the interaction between the B10 Tyr and the heme-bound NO, we examined the UV Raman spectrum of the B10 Tyr by subtracting the B10 mutant spectrum from the wild-type spectrum. It was found that, upon NO binding to the ferric protein, the Y(8a) mode of the B10 Tyr shifted from 1616 to 1622 cm(-1), confirming a direct interaction between the B10 Tyr and the heme-bound NO. Furthermore, the Y(8a) mode of the other two Tyr residues at positions 16 and 72 that are remote from the heme was also affected by NO binding, suggesting that NO binding to the distal site of the heme triggers a large-scale conformational change that propagates through the pre-F helix loop to the E and B helices. This large-scale conformational change triggered by NO binding may play an important role in regulating the ligand binding properties and/or the chemical reactivity of HbN.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated, tetrameric beta-chain from human adult hemoglobin

The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated beta-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D (1)H NMR in order to assess the extent of assignments allowed by (1)H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the beta-chain to the crystallographically characterized complexes that contains the beta-chain. The target residues are those that exhibit significant (>|0.2| ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 104 target residues ( approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than |0.2| ppm. These comprehensive assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the beta-chain in terms of the crystal coordinates of the beta-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated beta-chain relative to that in the intact R-state HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in the beta-subunit of HbCO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet beta-chain and that this approach should be similarly effective in HbA.     

X-ray crystallographic study of cyanide binding provides insights into the structure-function relationship for cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme. Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer. The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B. The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme. Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme. No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme.     

Human myeloperoxidase: structure of a cyanide complex and its interaction with bromide and thiocyanate substrates at 1.9 A resolution

The 1.9 A X-ray crystal structure of human myeloperoxidase complexed with cyanide (R = 0.175, R(free) = 0.215) indicates that cyanide binds to the heme iron with a bent Fe-C-N angle of approximately 157 degrees, and binding is accompanied by movement of the iron atom by 0.2 A into the porphyrin plane. The bent orientation of the cyanide allows the formation of three hydrogen bonds between its nitrogen atom and the distal histidine as well as two water molecules in the distal cavity. The 1.85 A X-ray crystal structure of an inhibitory complex with thiocyanate (R = 0.178, R(free) = 0.210) indicates replacement of chloride at a proximal helix halide binding site in addition to binding in the distal cavity in an orientation parallel with the heme. The thiocyanate replaces two water molecules in the distal cavity and is hydrogen bonded to Gln 91. The 1.9 A structures of the complexes formed by bromide (R = 0.215, R(free) = 0.270) and thiocyanate (R = 0.198, R(free) = 0.224) with the cyanide complex of myeloperoxidase show how the presence of bound cyanide alters the binding site for bromide in the distal heme cavity, while having little effect on thiocyanate binding. These results support a model for a single common binding site for halides and thiocyanate as substrates or as inhibitors near the delta-meso carbon of the porphyrin ring in myeloperoxidase.     

Solution 1H NMR study of the heme cavity and folding topology of the abbreviated chain 118-residue globin from the cyanobacterium Nostoc commune

The globin from the cyanobacterium Nostoc commune, abbreviated GlbN, which appears to serve as a part of a terminal oxidase rather than as a respiratory pigment, displays relatively normal O2 binding properties, despite the highly abbreviated polypeptide chain, (118 residues) relative to more conventional globins [Thorsteinsson, M. V. , Bevan, D. R., Potts, M., Dou, Y., Eich, R. F., Hargrove, M. S., Gibson, Q. H., and Olson, J. S. (1999) Biochemistry 38, 2117-2126]. The nature of the heme cavity and the general folding topology of this cyanoglobin were investigated by solution 1H NMR to establish the extent to which, and the manner in which, this compact globin adheres to the standard globin fold. This represents by far the smallest globin subjected to structural analysis. The paramagnetic cyanomet derivative was selected because its characteristically large magnetic anisotropy imparts significant dipolar shifts which both improve resolution to greatly facilitate assignments and serve as indicators of the folding topology of the globin. Identification of the axial His 70 and highly conserved Phe 35 (CD1) determined the absolute orientation of the heme and proximal His. Sequential assignments of four helical and one loop segments, which exhibit dipolar contacts to the heme and among each other, confirm the presence of well-conserved F, G, and H helices and the FG corner. The majority of the abbreviation of the chain relative to the more conventional length globins is accommodated in the A-D helices, of which the last is completely missing. The distal residue which provides a H-bond to bound ligand is identified as Gln 43, but the expected helical position E7 could not be confirmed. His 46, placed at position E10, is found to adopt alternate orientations into, and out of, the heme cavity depending on protonation state, suggesting the presence of a Bohr effect at low pH. It is shown that the dipolar shifts exhibited by backbone protons for the assigned residues conform well to those observed for other cyanomet globins and further support a conserved Mb fold. Perturbed medium-range dipolar contacts and the pH-independent backbone proton lability of the F helix are interpreted in terms of a holoprotein which is less stable than a conventional length globin.     

Nuclear-magnetic-resonance investigation of the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Molecular and electronic structure of the cyano-met derivative

The proton nuclear-magnetic-resonance spectra of the cyano-met complexes of the cooperative dimeric and tetrameric hemoglobins from the mollusk Scapharca inaequivalvis have been investigated and compared to those of other structurally characterized oxygen binding hemoproteins. For these proteins, cooperativity is displayed even in the homodimer and preliminary X-ray structural data reveal an unusual back-to-front assembly with intersubunit contacts involving the EF helices [Royer, W. E., Love, W. E. + Fenderson, F. F. (1985) Nature (Lond.) 316, 277-280]. The pattern of hyperfine shifts is very similar for the dimer and tetramer chains, but distinctly different from those of previously characterized low-spin, ferric heme proteins. Individual heme resonances are identified by reconstituting the protein with specifically deuterated hemes. While the axial interactions involving the proximal and distal histidines are very similar to that in myoglobins and other hemoglobins, both the heme contact shift pattern and the amino acid dipolar shift pattern reflect a significantly reduced asymmetry. The decreased spread of the non-cordinated amino acid signals is interpreted in terms of a rotation of the magnetic axes relative to those in myoglobin or other hemoglobins, rather than a change in the magnetic anisotropy. The decreased spread of the heme methyl contact shifts supports this conclusion and is consistent with an orientation of the proximal histidine with the imidazole ring rotated by about 30-40 degrees relative to that in other structurally characterized proteins. Although resonances associated with a complex pattern of alternate heme orientations can be detected immediately after reconstitution of the protein, the isolated protein was found to exhibit insignificant equilibrium heme rotational disorder.