Hydrogen bonding modulates binding of exogenous ligands in a myoglobin proximal cavity mutant.

In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. (1)H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.     

Hydrogen bonding of iron-coordinated histidine in heme proteins.

The hydrogen-bonding motifs of the proton on the N delta atom of iron-coordinated histidine residues in heme proteins have been classified into three categories: (1) Those in which the hydrogen-bond acceptor is either an amino acid residue (serine) directly adjacent to the histidine or a carbonyl group of the polypeptide chain less than five residues away from the histidine; (2) those in which the hydrogen-bonding acceptor is a carbonyl group of the polypeptide backbone associated with an amino acid residue 8 to 17 residues away from the histidine; and (3) those in which the hydrogen-bonding acceptor is an exogenous water molecule or an amino acid residue located far from the histidine in the amino acid sequence. Some biological functions are defined by this classification, whereas others span all classes.     

Electron-nuclear coupling in nitrosyl heme proteins and in nitrosyl ferrous and oxy cobaltous tetraphenylporphyrin complexes

Electron spin echo envelope modulation (ESEEM) spectroscopy has been used to study electron-nuclear interactions in the following isoelectronic S = 1/2 complexes: NO-FeII(TPP) (TPP = tetraphenylporphyrin) with and without axial nitrogenous base, nitrosylhemoglobin in R and T states, and O2-CoII(TPP) with and without axial base. Only the porphyrin pyrrole nitrogens contribute to the ESEEM of the 6-coordinate nitrosyl FeII(TPP) complexes, nitrosylhemoglobin (R-state), and the nitrosyl complexes of alpha and beta chains. Pyrrole nitrogens in the 5-coordinate complex NO-FeII(TPP) are coupled too weakly to unpaired spin and therefore do not contribute to the ESEEM. A partially saturated T-state nitrosylhemoglobin does not exhibit echo envelope modulations characteristic of 6-coordinate nitrosyl species, which confirms that the proximal imidazole bond to heme iron is disrupted. Study of 6-coordinate O2-CoII(TPP)(L) complexes (L = nitrogenous base) using 14N- and 15N-labeled ligands and porphyrins enabled a detailed analysis of coupling parameters for both pyrrole and axial nitrogens. The pyrrole 14N coupling frequencies are similar to those in NO-FeII(TPP)(L). The Fermi contact couplings for axially bound nitrogen, calculated from simulation of ESEEM spectra for a series of O2-CoII(TPP)(L) complexes (L = pyridine, 4-picoline, 4-cyanopyridine, 4-carboxypyridine, and 1-, 2-, and 4-methylimidazole) illustrate a trend toward stronger hyperfine interactions with weaker bases.     

Hydrogen bonding markedly reduces the pK of buried carboxyl groups in proteins

The ionizable groups in proteins with the lowest pKs are the carboxyl groups of aspartic acid side-chains. One of the lowest, pK=0.6, is observed for Asp76 in ribonuclease T1. This low pK appeared to result from hydrogen bonds to a water molecule and to the side-chains of Asn9, Tyr11, and Thr91. The results here confirm this by showing that the pK of Asp76 increases to 1.7 in N9A, to 4.0 in Y11F, to 4.2 in T91V, to 4.4 in N9A+Y11F, to 4.9 in N9A+T91V, to 5.9 in Y11F+T91V, and to 6.4 in the triple mutant: N9A+Y11F+T91V. In ribonuclease Sa, the lowest pK=2.4 for Asp33. This pK increases to 3.9 in T56A, which removes the hydrogen bond to Asp33, and to 4.4 in T56V, which removes the hydrogen bond and replaces the -OH group with a -CH(3) group. It is clear that hydrogen bonds are able to markedly lower the pK values of carboxyl groups in proteins. These same hydrogen bonds make large contributions to the conformational stability of the proteins. At pH 7, the stability of D76A ribonuclease T1 is 3.8 kcal mol(-1) less than wild-type, and the stability of D33A ribonuclease Sa is 4.1 kcal mol(-1) less than wild-type. There is a good correlation between the changes in the pK values and the changes in stability. The results suggest that the pK values for these buried carboxyl groups would be greater than 8 in the absence of hydrogen bonds, and that the hydrogen bonds and other interactions of the carboxyl groups contribute over 8 kcal mol(-1) to the stability.     

Hydrogen bonding in globular proteins.

A global census of the hydrogen bonds in 42 X-ray-elucidated proteins was taken and the following demographic trends identified: (1) Most hydrogen bonds are local, i.e. between partners that are close in sequence, the primary exception being hydrogen-bonded ion pairs. (2) Most hydrogen bonds are between backbone atoms in the protein, an average of 68%. (3) All proteins studied have extensive hydrogen-bonded secondary structure, an average of 82%. (4) Almost all backbone hydrogen bonds are within single elements of secondary structure. An approximate rule of thirds applies: slightly more than one-third (37%) form i----i--3 hydrogen bonds, almost one-third (32%) form i----i--4 hydrogen bonds, and slightly less than one-third (26%) reside in paired strands of beta-sheet. The remaining 5% are not wholly within an individual helix, turn or sheet. (5) Side-chain to backbone hydrogen bonds are clustered at helix-capping positions. (6) An extensive network of hydrogen bonds is present in helices. (7) To a close approximation, the total number of hydrogen bonds is a simple function of a protein's helix and sheet content. (8) A unique quantity, termed the reduced number of hydrogen bonds, is defined as the maximum number of hydrogen bonds possible when every donor:acceptor pair is constrained to be 1:1. This quantity scales linearly with chain length, with 0.71 reduced hydrogen bond per residue. Implications of these results for pathways of protein folding are discussed.     

Hydrogen bonding in DNA base complexes.

Experimental intermolecular frequencies in the DNA base complexes 1-methylthymine (1-MT) and cytosine monohydrate (CMH) are analyzed in terms of simple analytic interatomic potentials. Calculations with two different values for the constants of the nonbonded interactions are considered, and the hydrogen bond potentials are determined for each of these models. The observed frequencies in 1-MT are reasonably well described, although corresponding potentials are very different in the two models. The observed frequencies in CMH are less well described, although corresponding hydrogen bond potentials are similar in the two models. Hydration interactions are found to be important in CMH and the role of the water molecule is discussed. Possible reasons for the shortcomings of this simple analysis are considered.     

An infrared study of NO bonding to heme B and hemoglobin A. Evidence for inositol hexaphosphate induced cleavage of proximal histidine to iron bonds.

Five- and six-coordinate nitrosyl hemes have been prepared and their infrared, electron paramagnetic resonance (EPR), and visible-Soret spectra compared with the corresponding spectra for nitrosyl hemoglobin A (Hba-NO) determined both in the presence and the absence of inositol hexaphosphate (IHP). The five- and six-coordinate NO complexes prepared from either dipyridine or pyridine carbonyl protoheme dimethyl ester had N-O stretch bands (nuno) near 1675 and 1625 cm-1, respectively. These frequencies are sensitive to change in solvent (nuno decreased as the dipole moment of the solvent increased) and, with six-coordinate species, to changes in trans ligand. However, these solvent and trans ligand effects were small compared with the difference (ca. 50 cm-11) between five- and six -coordinate species. The nature of the trans ligand affected the relative proportions of the two...     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Solution 1H nuclear magnetic resonance determination of hydrogen bonding of the E10 (66) Arg side-chain to the bound ligand in Aplysia cyano-met myoglobin.

A combined one-dimensional nuclear Overhauser effect, paramagnetic-induced relaxation and two-dimensional sequence-specific 1H n.m.r. assignment of the spectrum of portions of the distal pocket of Aplysia cyano metMyoglobin (metMbCN) has been carried out in order to establish the presence and identity of distal residues in the heme pocket. In the absence of the usual distal E7 His in Aplysia Mb (E7 Val), the sequence-specific assignment of the E7 and E10 residues, together with their hyperfine shift patterns, relaxivities and dipolar connectivities to each other and the remainder of the E helix, reveal that the E10 Arg is turned into the pocket and hydrogen bonds to the bound cyanide group. We have previously found a similar rearrangement of the E10 Arg in Aplysia fluoro metMyoglobin, and the stabilizing effect of this residue was proposed to be responsible for the slow rate of cyanide dissociation from rapidly reduced ferrous Aplysia myoglobin. Based on the similar distal E7 His hydrogen-bonding interaction to the bound ligand in the crystal of sperm whale MbO2 and in solution of its cyano met complex, we propose that the E10 Arg similarly hydrogen bonds to the bound O2 in Aplysia MbO2 and accounts for its strong ligand binding and slow dissociation rate.     

Kinetics of carbon monoxide binding to monomeric hemoproteins. Role of the proximal histidine

The effect of pH on (i) the second-order rate constant for CO binding and (ii) the spectral properties of the deoxygenated derivative of several monomeric hemoproteins has been investigated in the pH range between 2.3 and 9.0. As in the case of 3-[1-imidazolyl]-propylamide monomethyl ester mesoheme, the rate constant for CO binding to sperm whale, horse, Dermochelys coriacea, Coryphaena hippurus, and Aplysia limacina myoglobins (the latter only in the presence of acetate/acetic acid mixture) increases, as the pH is lowered, to a value at least 1 order of magnitude higher than at pH 7.0. Such an effect is not observed in A. limacina myoglobin (in the absence of the acetate/acetic acid mixture) and Chironomus thummi thummi erythrocruorin. Moreover, the absorption spectrum, in the visible region, of the deoxy derivative of all these monomeric hemoproteins (with the exception of A. limacina myoglobin in the absence of the acetate/acetic acid mixture) undergoes a transition as the pH is lowered, an effect observed previously with 3-[1-imidazolyl]-propylamide monomethyl ester protoheme. On the basis of analogous spectroscopic and kinetic properties of chelated heme model compounds we attribute this behavior to the protonation of the N epsilon of the proximal imidazole involved in the bond with the iron atom. On the basis of this model the movement of the iron atom to the heme plane appears as a crucial step for CO binding, the activation free energy of the process amounting to approximately 2 kcal/mol.     

Hydrogen bonding of adenine derivatives to tyrosine side chain.

High resolution proton magnetic resonance measurements provide evidence for the formation of hydrogen-bonded complexes between 9-ethyladenine and p-cresol used as a model of tyrosine side chain in CDCl3. We have calculated the sum of the association constants corresponding to the three existing 1:1 complexes: K=6.3+/-0.15. By methylation of the amino group of adenine, we were able to calculate the ratio of the two strongest equilibrium constants K7/K1=1.6+/-0.3. Theoretical computations by the complete neglect of differential overlap (CNDO/2) method indicate that several hydrogen-bonded planar complexes can form between 9-methyladenine and phenol. The computed energy of the complexes with 6-dimethylamino adenine removes some ambiguity concerning the computed ratio of the association constants. Comparison of the calculated energies with free energies experimentally determined in organic solvent shows that despite the competition with CDCl3, which associates with both solute molecules, the preferential order of association is conserved. The small variations of charge density of adenine carbon atoms when complexed with phenol are in agreement with very small chemical shifts observed by 13C-nuclear magnetic resonance.     

The reactivity of alpha-hydroxyhaem and verdohaem bound to haem oxygenase-1 to dioxygen and sodium dithionite.

Recently we have shown that ferric alpha-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196-18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric alpha-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous alpha-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203-213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. alpha-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of alpha-hydroxyhaem with haem oxygenase may be small and a substantial amount of free alpha-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase.