Distal and proximal ligand interactions in heme proteins: correlations between C-O and Fe-C vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in C17O- and 13CO-labeled species

We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7----Val E7; His E7----Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C-O vibrational frequencies, v(C-O), and delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) and v(Fe-C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of pi-back-bonding from Fe d to CO pi* orbitals, as outlined previously [Li, X.-Y., & Spiro, T.G. (1988) J. Am. Chem. Soc. 110, 6024]. The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.     

13C shieldings, 18O isotope effects on 13C shieldings, and 57Fe-13C spin couplings of the Fe-C-O unit in superstructured hemoprotein models: Comparison with hemoproteins, C-O vibrational frequencies, and X-ray structural data

¹³C NMR spectra of several carbon monoxide (99.7% ¹³C and 11.8% ¹⁸O enriched) hemoprotein models with varying polar and steric effects of the distal organic superstructure and constraints of the proximal side are reported. This enables the ⁵⁷Fe-¹³C(O) coupling constants ( ), ¹³C shieldings ((¹³C)), and ¹⁸O isotope effects on¹³ C shieldings (¹¹³C(¹⁸O/¹⁶O)) to be measured and hence comparisons with hemoproteins, C-O vibrational frequencies and X-ray structural data to be made. Negative polar interactions in the binding pocket and inhibition of Fe//CO back-donation or positive distal polar interactions with amide NH groups appear to have little direct effect on couplings. Similarly, the axial hindered base 1,2-dimethylimidazole has a minor effect on the values despite higher rates of CO desorption being observed for such complexes. On the contrary,¹³ C shieldings vary widely and an excellent correlation was found between the infrared C-O vibrational frequencies ((C-O)) and¹³ C shieldings and a reasonable correlation with¹⁸ O isotope effects on ¹³C shieldings. This suggests that (¹³C), (C-O) and^{1 13} C(¹⁸O/¹⁶O) are accurate monitors of the multiple mechanisms by which steric and electronic interactions are released in superstructured heme model compounds. The ¹³C shieldings of heme models cover a 4.0 ppm range which is extended to 7.0 ppm when several HbCO and MbCO species at different pH values are included. The latter were found to obey a similar linear (¹³ (¹³C) versus (C-O) relationship, which proves that both heme models and heme proteins are homogeneous from the structural and electronic viewpoint. Our results suggest that (C-O), (¹³C) and ¹¹³C(¹⁸O/¹⁶O) measurements reflect similar interaction which is primarily the modulation of back-bonding from the Fe d to the CO * orbital by the distal pocket polar interactions. The lack of correlation between^{1 13} C(¹⁸O/¹⁶O) and crystallographic CO bond lengths (r(C-O)) reflects significant uncertainties in the X-ray determination of the carbon and oxygen positions.     

Nitric oxide interaction with insect nitrophorins and thoughts on the electron configuration of the {FeNO}6 complex

The nitrophorins are NO-carrying heme proteins that are found in the saliva of two species of blood-sucking insects, the kissing bug (Rhodnius prolixus) and the bedbug (Cimex lectularius). In both insects the NO is bound to the ferric form of the protein, which gives rise to Kds in the micromolar to nanomolar range, and thus upon injection of the saliva into the tissues of the victim the NO can dissociate to cause vasodilation and inhibition of platelet aggregation. The structures of the proteins from each of these insects are unique, and each has a large component of beta-sheet structure, which is unusual for heme proteins. While the Rhodnius nitrophorins increase the effectiveness of their NO-heme proteins by also binding histamine, secreted by the victim in response to the bite, to the heme, the Cimex nitrophorin does not bind histamine but rather binds two molecules of NO reversibly, one to the heme and the other to the cysteine thiolate which serves as the heme ligand in the absence of NO. This requires homolytic cleavage of the Fe-S-Cys bond, which produces an EPR-active Fe(II)-NO complex having the {FeNO}7 electron configuration. For the Rhodnius nitrophorins, the heme of the {FeNO}6 stable NO complex could have the limiting electron configurations Fe(III)-NO+ or Fe(II)-NO+. While vibrational spectroscopy suggests the latter and Mossbauer spectroscopy cannot differentiate between a purely diamagnetic Fe(II) center and a strongly antiferromagnetically coupled Fe(III)-NO* center, the strong ruffling of the heme (with alternate meso-carbons shifted significantly above and below the mean plane of the porphyrin, and concomitant shifts of the beta-pyrrole carbons above and below the mean plane of the porphyrin ring, to produce a very nonplanar porphyrin macrocycle) may suggest at least an important contribution of the latter. The strong ruffling would help to stabilize the (dxz, dyz)4(dxy)1 electron configuration of low-spin Fe(III) (but not low-spin Fe(II)), and the dxy orbital does not have correct symmetry for overlap with the half-filled pi* orbital of NO. This Fe(III)-NO* electron configuration would facilitate reversible dissociation of NO.     

Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

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

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

Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps

Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (nu(Fe)(-)(CO)) of the CO complex and Fe(3+)-S stretching mode (nu(Fe)(-)(S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The nu(Fe)(-)(CO) mode was upshifted from 477 to 485 and to 490 cm(-)(1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas nu(Fe)(-)(CO) was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of nu(Fe)(-)(S) by 5 cm(-)(1) compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe(4+)=O porphyrin(+)(*)) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.     

Functional consequences of the creation of an Asp-His-Fe triad in a 3/3 globin

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.     

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.     

Differential additions to the myoglobin prosthetic heme group. Oxidative gamma-meso substitution by alkylhydrazines.

The oxidative reaction of equine myoglobin with alkylhydrazines results primarily in introduction of the alkyl group at the sterically hindered gamma-meso position. The gamma-meso adducts formed with ethyl- and n-butylhydrazine have been isolated and unambiguously identified. With high pressure liquid chromatography, evidence for the formation of similar adducts with methyl- and n-propylhydrazine but not tert-butyl-, 2,2,2-trifluoroethyl-, or 2-phenylethylhydrazine has also been obtained. The gamma regiospecificity of the reaction of myoglobin with alkylhydrazines contrasts with the delta meso regiospecificity in the alkylation of peroxidases. Addition to the porphyrin vinyl groups is not detected, but N-alkylheme adducts appear to be formed in very low yield. Cofactor studies establish that H2O2 is absolutely required for meso heme alkylation and EPR/spin trapping studies show that alkyl free radicals are the probable alkylating species. In contrast, the reductive reaction of sperm whale myoglobin with CBrCl3 results in addition of the CCl3.radical to the 2-vinyl moiety of the heme group (Osawa, Y., Highet, R. J., Murphy, C. M., Cotter. R.J., and Pohl, L.R. (1989) J. Am. Chem. Soc. 111, 4462-4467). Carbon radicals thus apparently add to different sites of the myoglobin prosthetic group under reductive and oxidative conditions, presumably because of differences in the oxidation state of the heme and/or the intrinsic reactivities of alkyl and polyhaloalkyl radicals.     

Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme

Macrophage-generated oxygen- and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis 'truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for approximately 28 A through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.     

A comparative resonance Raman analysis of heme-binding PAS domains: heme iron coordination structures of the BjFixL, AxPDEA1, EcDos, and MtDos proteins

The heme-PAS is a specialized domain with which a broad class of signal-transducing heme proteins detect physiological heme ligands. Such domains exhibit a wide range of ligand binding parameters, yet they are all expected to feature an alpha-beta heme binding fold and a predominantly hydrophobic heme distal pocket without a distal histidine. We have compared, for the first time, the resonance Raman spectra of several heme-PASs: the heme-binding domains of Bradyrhizobium japonicum FixL, Escherichia coli Dos, Acetobacter xylinum PDEA1, and Methanobacterium thermoautotrophicum Dos. In all cases, the nu(Fe)-(CO) and nu(C-O) values of the carbonmonoxy forms were consistent with coordination of the heme iron to histidine on the proximal side and binding of the CO without electrostatic interaction with the heme distal pocket. EcDos was unusual in having predominantly hexacoordinate heme iron in the deoxy and met forms. Despite an evident lack of CO interaction with the EcDos heme pocket, relatively low Fe-O(2) (562 cm(-1)) and N-O (1576 cm(-1)) stretching frequencies indicated that strong polar interactions with that heme distal pocket are possible for highly bent ligands such as O(2) or NO. None of the newly studied NO adducts exhibited evidence of the Fe-His rupture and pentacoordination previously noted for Sinorhizobium meliloti FixL. A low Fe-His stretching frequency, formerly interpreted as a strained Fe-His bond, and the slow association of O(2) with S. meliloti FixL failed to correlate with the newly studied proteins having low association rate or low equilibrium association constants for binding of O(2). We conclude that although heme-PASs share some features, they represent distinct signal transduction mechanisms.     

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

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

Mixed regioselectivity in the Arg-177 mutants of Corynebacterium diphtheriae heme oxygenase as a consequence of in-plane heme disorder

It has been reported that the R183E and R183D mutants of rat heme oxygenase-1 (r-HO-1) produce approximately 30% delta-biliverdin [Zhou, H., et al. (2000) J. Am. Chem. Soc. 122, 8311-8312]. Two plausible mechanisms were proposed to explain the observations. (a) Electrostatic repulsion between E183 (D183) and one of the heme propionates forces the heme to rotate, thereby placing the delta-meso carbon in a position that is susceptible to oxidation. (b) Rearrangement of the distal pocket structure is triggered by the formation of a hydrogen bond between E183 (D183) and K179. A change in the pK(a) for the Fe(III)-H(2)O to Fe(III)-OH transition of the mutants was interpreted to be consistent with rearrangement of the hydrogen bond network in the distal pocket. The large similarities between the high-frequency portion of the (1)H NMR spectra corresponding to the wild type and R183E and R183D mutants were interpreted to indicate that the heme in the mutants is not rotated to a significant extent. We have re-examined this issue by studying the corresponding R177 mutants in heme oxygenase from Corynebacterium diphtheriae (cd-HO). Replacing R177 with E or D results in the formation of approximately 55% alpha- and 45% delta-biliverdin, whereas the R177A mutant retains alpha-regioselectivity. In addition, the K13N/Y130F/R177A triple mutant catalyzed the formation of 60% delta- and 40% alpha-biliverdin, while single mutants K13N and Y130F did not appreciably change the regioselectivity of the reaction. The pK(a) of the Fe(III)-H(2)O to Fe(III)-OH transition in wild-type cd-HO is 9.1, and those of the R177E, R177D, R177A, and K13N/Y130F/R177A mutants are 9.4, 9.5, 9.2, and 8.0, respectively. Thus, no obvious correlation exists between the changes in pK(a) and the altered regioselectivity. NMR spectroscopic studies conducted with the R177D and R177E mutants of cd-HO revealed the presence of three heme isomers: a major (M) and a minor (m) heme orientational isomer related by a 180 degrees rotation about the alpha-gamma meso axis and an alternative seating (m') which is related to m by an 85 degrees in-plane rotation of the macrocycle. The in-plane rotation of m to acquire conformation m' is triggered by electrostatic repulsion between the side chains of D or E at position 177 and heme propionate-6. As a consequence, the delta-meso carbon in m' is placed in the position occupied by the alpha-meso carbon in m, where it is susceptible to hydroxylation and subsequent formation of delta-biliverdin.     

Structure and ligand binding properties of leucine 29(B10) mutants of human myoglobin.

Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29 (B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta meso carbon of the heme group to the one pointing toward the alpha meso carbon atom, presumably filling the space left by removal of the delta 2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)----Ala and Leu104 (G5)----Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.     

Resonance Raman spectra of bovine liver catalase compound II. Similarity of the heme environment to horseradish peroxidase compound II

Resonance Raman spectroscopy has been used to investigate the structure and environment of the heme group in bovine liver catalase compound II. Both Soret- and Q-band excitation have been employed to observe and assign the skeletal stretching frequencies of the porphyrin ring. The oxidation state marker band v4 increases in frequency from 1373 cm-1 in ferricatalase to 1375 cm-1 in compound II, consistent with oxidation of the iron atom to the Fe(IV) state. Oxidation of five-coordinate, high-spin ferricatalase to compound II is accompanied by a marked increase of the porphyrin core marker frequencies that is consistent with a six-coordinate low-spin state with a contracted core. An Fe(IV) = O stretching band is observed at 775 cm-1 for compound II at neutral pH, indicating that there is an oxo ligand at the sixth site. At alkaline pH, the Fe(IV) = O stretching band shifts to 786 cm-1 in response to a heme-linked ionization that is attributed to the distal His-74 residue. Experiments carried out in H218O show that the oxo ligand of compound II exchanges with bulk water at neutral pH, but not at alkaline pH. This is essentially the same behavior exhibited by horseradish peroxidase compound II and the exchange reaction at neutral pH for both enzymes is attributed to acid/base catalysis by a distal His residue that is believed to be hydrogen-bonded to the oxo ligand. Thus, the structure and environment of the heme group of the compound II species of catalase and horseradish peroxidase are very similar. This indicates that the marked differences in their reactivities as oxidants are probably due to the manner in which the protein controls access of substrates to the heme group.     

Critical role of the heme axial ligand, Met95, in locking catalysis of the phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O(2) or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O(2) molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O(2) complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O(2). In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O(2), CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.     

Electron-nuclear double resonance studies of oxidized Escherichia coli sulfite reductase: 1H, 14N, and 57Fe measurements

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the bridged siroheme--[Fe4S4] cluster that forms the catalytically active center of the oxidized hemoprotein subunit (SiRo) of Escherichia coli NADPH-sulfite reductase. The siroheme 57Fe hyperfine coupling (Az = 27.6 MHz, Ay = 26.8 MHz) is similar to that of other high-spin heme systems (A approximately equal to 27 MHz). Bonding parameters obtained from the 14N hyperfine coupling constants of the siroheme pyrrole nitrogens are consistent with a model of a nonplanar pi system of reduced aromaticity. The absence of hyperfine coupling to the 14N of an axial ligand, such as is observed for the histidine 14N of metmyoglobin (Az = 11.55 MHz), rules out the possibility that imidazolate acts as the bridge between the siroheme and the [Fe4S4] cluster. Proton ENDOR of the deuterium-exchanged protein indicates that H2O does not function as a sixth axial ligand and suggests that the ferrisiroheme is five-coordinate. 57Fe ENDOR measurements confirm the results of M?ssbauer spectroscopy for the [Fe4S4] cluster. They also disclose a slight anisotropy of the cluster 57Fe coupling that may be associated with the mechanism by which the siroheme and cluster spins are coupled.     

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

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

Role of spin state on the geometry and nuclear quadrupole resonance parameters in hemin complex

Theoretical calculations of structural parameters, 57Fe, 14N and 17 O electric field gradient (EFG) tensors for full size-hemin group have been carried out using density functional theory. These calculations are intended to shed light on the difference between the geometry parameters, nuclear quadrupole coupling constants (QCC), and asymmetry parameters (eta Q) found in three spin states of hemin; doublet, quartet and sextet. The optimization results reveal a significant change for propionic groups and porphyrin plane in different spin states. It is found that all principal components of EFG tensor at the iron site are sensitive to electronic and geometry structures. A relationship between the EFG tensor at the 14N and 17 O sites and the spin state of hemin complex is also detected.