Studies of the electron-nuclear coupling between Fe(III) and 14N in cytochrome P-450 and in a series of low spin heme compounds.

We have observed the nuclear modulation pattern in the envelope of electron spin echoes for various low spin paramagnetic heme proteins including cytochrome c, myoglobin hydroxide, myoglobin mercaptoethanol, and cytochrome P-450, using the three-pulse-stimulated echo method. We have also carried out similar experiments with model compounds containing either [14N]- or [15N]imidazole. In many of the compounds studied, we have been able to identify the nuclear modulation effects arising from 14N of the porphyrin ring and have been able to characterize and interpret the modulation effects due to 14N of various nitrogenous axial ligands. We have found that the heme of low spin ferric cytochrome P-450 is coordinated to a nitrogenous ligand, probably imidazole. We have also demonstrated that the remote 14N of the imidazole ligand in a [14N]imidazole-heme-NO-model compound is coupled differently than in myoglobin nitroxide, demonstrating the direct effect of the protein of metal ligand bonding.     

Low-temperature electron paramagnetic resonance study of the ferricytochrome c-cardiolipin complex

The electron paramagnetic resonance spectrum of the ferricytochrome c complex with cardiolipin was observed at temperatures below 20 K. For the low-spin iron(III) heme system complexed with the negatively charged lipid, the tetragonal and rhombic ligand field parameters (delta/lambda = 3.58, V/lambda = 1.82) differ significantly from those (delta/lambda = 2.53, V/lambda = 1.49) of the free ferricytochrome c sample. The g values of the complex (gx = 1.54 +/- 0.02, gy = 2.26 +/- 0.01, gz = 3.02 +/- 0.01) are compared to the values for free ferricytochrome c (gx = 1.25 +/- 0.02, gy = 2.25 +/- 0.01, gz = 3.04 +/- 0.01). Spectral alterations are interpreted in terms of the ligand field changes induced within the heme group by association with the negatively charged phosphoglyceride.     

A photolysis-triggered heme ligand switch in H93G myoglobin

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in < 5 micros. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase.     

Cyanate binding to the ferric heme octapeptide from cytochrome c. A model for anion binding to high spin ferric hemoproteins

Equilibrium constants for the binding of cyanate to the ferric heme c octapeptide in 50% ethylene glycol, 50% aqueous buffer were measured spectrophotometrically. Equilibrium constants measured at several temperatures from -20 degrees C to 0 degrees C exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta Ho = -1.3 X 10(3) +/- 0.9 X 10(3) J/mol (-3.1 X 10(2) +/- 2 X 10(2) cal/mol), delta So = -3 +/- 3 J/K X mol (-0.6 +/- 0.8 cal/K X mol). The equilibrium constant for cyanate binding at 25 degrees C and pH 7.4 is 1.21 which is approximately 2 to 3 orders of magnitude lower than that observed for cyanate binding to methemoglobin and metmyoglobin. Krel, the ratio of the hemoprotein to model heme octapeptide binding constants, for NCO- is smaller than Krel for N3- suggesting that hydrogen bonding between the terminal ligand atoms and the distal histidine in hemoglobin and myoglobin does not contribute to the increased protein ligand stabilization observed for these anions relative to the model. A donor-acceptor interaction between the distal histidine and the electrophilic middle atoms of these bound ligands is proposed.     

Spectroscopic properties of ferrous heme complexes of sterically hindered ligands.

Mesoheme IX complexes of sterically hindered ligands 2-methylimidazole, tert-butylamine and 2-methylpyridine in aqueous glycerol solutions are characterized by broad visible absorption spectra at ambient temperature exhibiting close similarities to high-spin ferrous hemeproteins. Spectrophotometric titrations of mesoheme IX with these ligands indicate well-defined equilibria for 2-methylimidazole and tert-butylamine corresponding to the formation of penta-coordinate strong-field ligand complexes. Variable temperature spectra of these complexes from ambient to 77 degrees K exhibit a change to hemochrome spectra characteristic of the low-spin unhindered ligand complexes. Corresponding changes in the visible spectra are not observed for the high-spin hemeproteins deoxymyoglobin, horse-radish peroxidase and cytochrome ?. The appropriate utilization of these hindered ligand heme complexes as model systems for high-spin ferrous hemeproteins has been discussed.     

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

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.     

Spectroscopic and equilibrium studies of ligand and organic substrate binding to indolamine 2,3-dioxygenase

The binding of a number of ligands to the heme protein indolamine 2,3-dioxygenase has been examined with UV-visible absorption and with natural and magnetic circular dichroism spectroscopy. Relatively large ligands (e.g., norharman) which do not readily form complexes with myoglobin and horseradish peroxidase (HRP) can bind to the dioxygenase. Except for only a few cases (e.g., 4-phenylimidazole) for the ferric dioxygenase, a direct competition for the enzyme rarely occurs between the substrate L-tryptophan (Trp) and the ligands examined. L-Trp and small heme ligands (CN-,N3-,F-) markedly enhance the affinity of each other for the ferric enzyme in a reciprocal manner, exhibiting positive cooperativity. For the ferrous enzyme, L-Trp exerts negative cooperativity with some ligands such as imidazoles, alkyl isocyanides, and CO binding to the enzyme. This likely reflects the proximity of the Trp binding site to the heme iron. Other indolamine substrates also exert similar but smaller cooperative effects on the binding of azide or ethyl isocyanide. The pH dependence of the ligand affinity of the dioxygenase is similar to that of myoglobin rather than that of HRP. These results suggest that indolamine 2,3-dioxygenase has the active-site heme pocket whose environmental structure is similar to, but whose size is considerably larger than, that of myoglobin, a typical O2-binding heme protein. Although the L-Trp affinity of the ferric cyanide and ferrous CO enzyme varies only slightly between pH 5.5 and 9.5, the unligated ferric and ferrous enzymes have considerably higher affinity for L-Trp at alkaline pH than at acidic pH. L-Trp binding to the ferrous dioxygenase is affected by an ionizable residue with a pKa value of 7.3.     

Resonance Raman enhancement of the Mn-N-O bending mode in nitrosyl manganese "strapped" and "open" heme complexes

Resonance Raman spectra of the MnII-NO moiety in synthetic nitrosyl manganese heme complexes with and without steric hindrance are reported. The "strapped" hemes having a hydrocarbon strap (variable length) across one face of the heme hinder the perpendicular bonding of a linear ligand. These complexes were employed to investigate the effects of ligand distortion (primarily tilting) on Mn-NO stretching, Mn-N-O bending, and N-O stretching modes. It is demonstrated that ligand distortion in the MnII-NO system is a valid mechanism for causing the resonance enhancement of the Mn-N-O bending mode, similar to that observed in the FeII-CO system (Yu, N.-T., E. A. Kerr, B. Ward, and C. K. Chang. 1983. Biochemistry. 22:4534-4540). More interesting is the observation of the delta(Mn-N-O) enhancement caused by the tilting of the trans Mn-N epsilon bond in the "open" heme complexes (e.g., heme-5 and proto-1X dimethylester) with 1,2-dimethylimidazole or piperidine as a base. The nu(Mn-NO) and nu(N-O) modes exhibit an increase and a decrease, respectively, as the strap length decreases (hence the steric hindrance increases). Both nu(Mn-NO) and nu(N-O) frequencies are insensitive to the strength of the trans base. The results from "strapped" and "open" model heme systems imply that the Mn-N-O geometry is essentially linear and perpendicular in the nitrosyl complexes of monomeric manganese insect hemoglobin CTT IV and sperm whale myoglobin. The unusually low nu(N-O) frequency in the manganese myoglobin complex may be caused by the distal histidine-NO interaction. The delta(Mn-N-O) enhancement in both nitrosyl manganese CTT IV and nitrosyl manganese myoglobin may be caused by a tilting of the Mn"-Nf (proximal histidine) bond.     

Resonance Raman studies of CO and O2 binding to elephant myoglobin (distal His(E7)----Gln)

Carbon monoxide and dioxygen were employed as resonance Raman-visible ligands for probing the nature of the heme-binding site in elephant myoglobin, which has glutamine in the distal position (E7) instead of the usual histidine. The distal histidine (E7) residue has been thought to be responsible for weakening carbon monoxide binding to hemoproteins. It is of interest to see how the His(E7)----Gln replacement affects such parameters as nu(Fe-N epsilon), nu(Fe-CO), delta(Fe-C-O), nu(C-O), delta(Fe-O-O), and nu(O-O) vibrational frequencies and relative intensities. Elephant myoglobin has a CO affinity approximately 6 times higher than that for human/sperm whale myoglobin (Mb). If this enhanced affinity were solely due to the removal of some of the steric hindrance that normally tilts the CO off the heme axis, one would expect the nu(Fe-CO) frequency to decrease and the nu(C-O) frequency to increase relative to the corresponding values in sperm whale Mb. However, the opposite was found. In addition, strong enhancement of the Fe-C-O bending mode was observed. These results suggest that the Fe-C-O linkage remains distorted. In elephant Mb, new interactions resulting from the conformational change accompanying ligand binding may be responsible for the increased CO binding. Similar spectra were obtained for elephant and sperm whale oxymyoglobin. This suggests that the interactions of bound O2 are not markedly affected by the glutamine replacement.     

Novel heme ligation in a c-type cytochrome involved in thiosulfate oxidation: EPR and MCD of SoxAX from Rhodovulum sulfidophilum

The SoxAX complex of the bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that plays an essential role in photosynthetic thiosulfate and sulfide oxidation. The three heme sites of SoxAX have been analyzed using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies. Heme-3 in the ferric state is characterized by a Large g(max) EPR signal and has histidine and methionine axial heme iron ligands which are retained on reduction to the ferrous state. Hemes-1 and -2 both have thiolate plus nitrogenous ligand sets in the ferric state and give rise to rhombic EPR spectra. Heme-1, whose ligands derive from cysteinate and histidine residues, remains ferric in the presence of dithionite ion. Ferric heme-2 exists with a preparation-dependent mixture of two different ligand sets, one being cysteinate/histidine, the other an unidentified pair with a weaker crystal-field strength. Upon reduction of the SoxAX complex with dithionite, a change occurs in the ligands of heme-2 in which the thiolate is either protonated or replaced by an unidentified ligand. Sequence analysis places the histidine/methionine-coordinated heme in SoxX and the thiolate-liganded hemes in SoxA. SoxAX is the first naturally occurring c-type cytochrome in which a thiolate-coordinated heme has been identified.     

Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and 1H NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E0') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pKa values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S0'rc and Delta H0'rc) for the various protein conformers [low- and high-T native (N1 and N2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees ' values very similar to those of the mammalian proteins; (ii) the N1 --> N2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7. 5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pKa for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c2.     

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.     

The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.     

Investigations of the myoglobin cavity mutant H93G with unnatural imidazole proximal ligands as a modular peroxide O-O bond cleavage model system

A general inability to elucidate extensive variations in the electronic characteristics of proximal heme iron ligands in heme proteins has hampered efforts to obtain a clear understanding of the role of the proximal heme iron ligand in the activation of oxygen and peroxide. The disadvantage of the frequently applied site-directed mutagenesis technique is that it is limited by the range of natural ligands available within the genetic code. The myoglobin cavity mutant H93G [Barrick, D. (1994) Biochemistry 33, 6546-6554] has its proximal histidine ligand replaced with glycine, a mutation which leaves an open cavity capable of accommodating a variety of unnatural potential proximal ligands. We have carried out investigations of the effect of changing the electron donor characteristics of a variety of substituted imidazole proximal ligands on the rate of formation of myoglobin compound II and identified a correlation between the substituted imidazole N-3 pK(a) (which provides a measure of the electron donor ability of N-3) and the apparent rate of formation of compound II. A similar rate dependence correlation is not observed upon binding of azide. This finding indicates that O-O bond cleavage and not the preceding peroxide binding step is being influenced by the electron donor characteristics of the substituted imidazole ligands. The proximal ligand effects are clearly visible, but their overall magnitude is quite low (1.7-fold increase in the O-O bond cleavage rate per pK(a) unit). This appears to provide support for recent commentaries which concluded that the partial ionization of the proximal histidine ligand in typical heme peroxidases may not be enough of an influence to provide a mechanistically critical push effect [Poulos, T. L. (1996) JBIC, J. Biol. Inorg. Chem. 1, 356-359]. Further attempts were made to define the mechanism of the influence of N-3 pK(a) on O-O bond cleavage by using peracetic acid and cumene hydroperoxide as mechanistic probes. The observation of heme destruction in these reactions indicates that displacement of the proximal imidazole ligands by peracetic acid or cumene hydroperoxide has occurred. A combination mutation (H64D/H93G) was prepared with the objective of observing compound I of H64D/H93G with substituted imidazoles as proximal ligands upon reaction with H(2)O(2). This double mutant was found to simultaneously bind imidazole to both axial positions, an arrangement which prevents a reaction with H(2)O(2).     

H93G myoglobin cavity mutant as versatile template for modeling heme proteins: magnetic circular dichroism studies of thiolate- and imidazole-ligated complexes

Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

The first enantioselective synthesis of the amavadin ligand and its complexation to vanadium

The ligand of the naturally occurring vanadium compound amavadin found in Amanita muscaria, (2S, 2'S)-N-hydroxyimino-2,2'-dipropionic acid (1), was synthesized stereoselectively in two steps with 43% overall yield. After complexation of this ligand to vanadyl acetate, amavadin was isolated in quantitative yield. Due to the chirality at vanadium amavadin consists of a mixture of delta and lambda diastereoisomers. Directly after its synthesis, the delta to lambda ratio of amavadin is 2.27 and it decreases to 0.80 after equilibrium has been reached. During this epimerization the optical rotation for V[(2S,2'S)-N-hydroxyimino-(2,2')-dipropionate]2 (=amavadin) changes from [alpha](D)25 = +36 degrees to +114.0 degrees (c = 0.5, H2O). For V[(2R,2'R)-N-hydroxyimino-(2,2')-dipropionate] the optical rotation changes from [alpha](D)25 = -36 degrees to -113.2 degrees (c = 0.5, H2O).