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)     

Site-directed mutagenesis of the heme axial ligands in the hemoflavoenzyme cellobiose dehydrogenase

Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium is an extracellular 90-kDa hemoflavoenzyme, organized into an N-terminal heme domain and a C-terminal flavin domain. The amino acid residues Met65 and His114 or His163 were suggested to be heme iron ligands. Mutations of these residues were made and mutant proteins were characterized. H114A mutant cultures produce a stable hemoflavoenzyme with spectral and kinetic characteristics similar to those of wild-type CDH. The M65A and H163A transformants secrete a 90-kDa hemoflavoenzyme, which oxidizes cellobiose in the presence of 2,6-dichlorophenol-indophenol (DCPIP), but is unable to reduce cytochrome c. The heme domains of the M65A and H163A CDH variants are, however, unstable and susceptible to degradation, both yielding a 70-kDa cellobiose-oxidizing flavoenzyme. The spectral and kinetic characteristics of these truncated variants suggest that they contain only their respective flavin domains. The yield of the 90-kDa proteins was low and the proteins could not be purified to homogeneity; however, absorption spectra indicate that the 90-kDa proteins do contain the heme domain. Like the truncated flavoenzymes, the 90-kDa variants reduce DCPIP but are unable to transfer electrons to cytochrome c, in contrast to wild-type CDH. These findings suggest that H163 and M65 are the axial heme ligands and that both ligands are required for the reactivity and structural integrity of the heme domain.     

Replacement of putative axial ligands of heme iron in yeast cytochrome c1 by site-directed mutagenesis

The His-44 and Met-164 residues of yeast cytochrome c1 are evolutionally conserved and regarded as heme axial ligands bonding to the fifth and sixth coordination sites of the heme iron, which is directly involved in the electron transfer mechanism. Oligonucleotide-directed mutagenesis was used to generate mutant forms of cytochrome c1 of yeast having amino acid replacements of the putative axial ligands of the heme iron. When a cytochrome c1-deficiency yeast strain was transformed with a gene encoding the Phe-44, Tyr-44, Leu-164, or Lys-164 protein, none of these transformants could grow on the non-fermentable carbon source. These results suggest that the His-44 and Met-164 residues have a critical role in the function of cytochrome c1 in vivo, most probably as axial ligands of the heme iron. Further analysis revealed that the mutant yeast cells with the Phe-44, Tyr-44, or Leu-164 protein lacked the characteristic difference spectroscopic signal of cytochrome c1. However, in the Lys-164 mutant cells, partial recovery of the cytochrome c1 signal was observed. Moreover, the Lys-164 protein retained a low but significant level of succinate-cytochrome c reductase activity in vitro. The possibility that the nitrogen of Lys-164 served as the sixth heme ligand is discussed in comparison with cytochrome f of a photosynthetic electron-transfer complex, in which lysine has been proposed to be the sixth ligand.     

Alteration of the specificity of restriction endonucleases in the presence of organic solvents

The specificity of XbaI, SalI, HhaI, PstI, BamHI and SstI is relaxed in the presence of an organic solvent, such as 20% glycerol or 8% dimethylsulfoxide (DMSO). This alteration, very pronounced in some cases, requires an excess of enzyme, varies from one kind of enzyme to another and is highly dependent on the pH, the ionic strength, the nature of the metallic cofactor and/or the presence of a second organic solvent. Preliminary data concerning XbaI and BamHI used under conditions where the relaxation of specificity is moderate, suggest that some of the new ("pseudo") sites correspond to shortened sequences derived from the normal recognition sequence cleaved under the standard conditions of the reaction.     

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley & Sons, Inc.     

Location of heme axial ligands in the cytochrome d terminal oxidase complex of Escherichia coli determined by site-directed mutagenesis

The cytochrome d terminal oxidase complex is one of two terminal oxidases which are components of the aerobic respiratory chain of Escherichia coli. This membrane-bound enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Enzyme turnover generates proton and voltage gradients across the bilayer. The oxidase is a heterodimer containing 2 mol of protoheme IX and 1 or 2 mol of heme d per mol of complex. To explain the functional properties of the enzyme, a simple model has been proposed in which it is speculated that the heme prosthetic groups define two separate active sites on opposite sides of the membrane at which the oxidation of quinol and the reduction of water, respectively, are catalyzed. This paper represents an initial effort to define the axial ligands of each of the three or four hemes within the amino acid sequence of the oxidase subunits. Each of the 10 histidine residues has been altered by site-directed mutagenesis with the expectation that histidine residues are likely candidates for heme ligands. Eight of the 10 histidine residues are not essential for enzyme activity, and 2 appear to function as heme axial ligands. Histidine 186 in subunit I is required for the cytochrome b558 component of the enzyme. This residue is likely to be located near the periplasmic surface of the membrane. Histidine 19, near the amino terminus of subunit I also appears to be a heme ligand. It is concluded that two of the four or five expected heme axial ligands have been tentatively identified, although further work is required to confirm these conclusions. A minimum of two additional axial ligands must be residues other than histidine.     

A cooperative oxygen binding hemoglobin from Mycobacterium tuberculosis. Stabilization of heme ligands by a distal tyrosine residue

The homodimeric hemoglobin (HbN) from Mycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O(2), CO, and OH(-) derivatives of the wild type protein and the B10 Tyr --> Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.     

Structural and electronic characterization of heme moiety in oxygenated hemoproteins by using XANES spectroscopy.

Iron K-edge X-ray absorption near edge structure (XANES) spectra were measured for oxy-forms of cytochrome P-450cam (P-450cam), horseradish peroxidase (HRP) and myoglobin (Mb) by using Synchrotoron Radiation of Photon Factory (Tsukuba). A pronounced 1s-4p transition and some fine structures were well-resolved in the spectra obtained. Comparing the spectra, the features at the fine structures termed P, C and D, were similar among the three hemoproteins, suggesting a similar site-symmetry around the heme iron and the same Fe-O-O bond angle (about 115 degrees). On the other hand, absorption features at the edge region (7115-7135 eV) were slightly but significantly different from one another; the absorption intensity at 7115-7125 eV region increased in the order of Mb, HRP and P-450cam, while that at 7125-7135 eV decreased in the same order. A similar absorption feature was also obtained with their deoxy (ferrous high spin) forms. We assumed that the absorption at the lower energy region (7115-7125 eV) reflects the pi-character in the Fe-ligand bond, whereas that at the higher energy region (7125-7135 eV) does the sigma-character, on the basis of the previous and comprehensive studies of the XANES spectroscopy of the adsorbed molecules on the metal surface (McGovern et al. (1989) Handbook on Synchrotoron Radiation, Vol. 2, pp. 467-539). According to our assumption, our XANES results indicated that the pi-character of the Fe-ligand bond increases in the order of Mb, HRP and P-450cam, and that the pi-electron of the thiolate S- in P-450cam is donated to the Fe-O-O moiety, most probably to the antibonding pi* orbital of O2. Such an interpretation is consistent with the experimental findings or data accumulated so far by other methods, such as the resonance Raman spectroscopy.     

Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (SDH) plays a key role in energy generation by coupling the oxidation of succinate to the reduction of ubiquinone in the mitochondrial electron transport chain. The Saccharomyces cerevisiae SDH is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits. We systematically mutated all the histidine and cysteine residues in Sdh3p and Sdh4p to identify the residues involved in axial heme ligation. The mutants were characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction, for heme b content, and for heme spectral properties. Mutation of Sdh3p His-46 or His-113 leads to a marked reduction in the catalytic efficiency of the enzyme for quinone reduction, suggesting that these residues form part of a quinone-binding site. We identified Sdh3p His-106 and Sdh4p Cys-78 as the most probable axial ligands for cytochrome b(562). Replacement of His-106 or Cys-78 with an alanine residue leads to a marked reduction in cytochrome b(562) content and to altered heme spectral characteristics that are consistent with a direct perturbation of heme b environment. This is the first identification of a cysteine residue serving as an axial ligand for heme b in the SDH family of enzymes. Loss of cytochrome b(562) has no effect on enzyme assembly and quinone reduction; the role of the heme in enzyme structure and function is discussed.     

Heterogeneity of the isolated subunits of the fetal and adult human hemoglobin in solution, detected by XANES spectroscopy

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.     

Redox infrared markers of the heme and axial ligands in microperoxidase: bases for the analysis of c-type cytochromes

Structural changes accompanying the change in the redox state of microperoxidase-8 (MP8), the heme-octapeptide obtained from cytochrome c, and its complexes with (methyl)imidazole ligands were studied by electrochemically induced Fourier transform IR (FTIR) difference spectroscopy. To correlate with confidence IR modes with a specific electronic state of the iron, we used UV-vis and electron paramagnetic resonance spectroscopy to define precisely the heme spin state in the samples at the millimolar concentration of MP8 required for FTIR difference spectroscopy. We identified four intense redox-sensitive IR heme markers, nu38 at 1,569 cm(-1) (ox)/1,554 cm(-1) (red), nu42 at 1,264 cm(-1) (ox)/1,242 cm(-1) (red), nu43 at 1,146 cm(-1) (ox), and nu44 at 1,124-1,128 cm(-1) (ox). The intensity of nu42 and nu43 was clearly enhanced for low-spin imidazole-MP8 complexes, while that of nu44 increased for high-spin MP8. These modes can thus be used as IR markers of the iron spin state in MP8 and related c-type cytochromes. Moreover, one redox-sensitive band at 1,044 cm(-1) (red) is attributed to an IR marker specific of c-type hemes, possibly the delta(CbH3)(2,4) heme mode. Other redox-sensitive IR bands were assigned to the MP8 peptide backbone and to the fifth and sixth axial heme ligands. The distinct IR frequencies for imidazole (1,075 cm(-1)) and histidine (1,105 cm(-1)) side chains in the imidazole-MP8 complex allowed us to provide the first direct determination of their pKa at pH 9 and 12, respectively.     

Structure and variability of human chromosomes analyzed by recent techniques

Summary Besides the AT-specific fluorochromes, GC-specific fluorescent antibiotics are now available for chromosomal analysis. Chromosomal bands represent large accumulation of DNA sequences with similar AT:GC ratio. These uniform differences from the mean AT:GC ratio in the bands can be explained only by at least partial repetition of short DNA sequences in these regions. By comparison of various staining techniques more information also on the constitutive heterochromatin in man becomes available. The human NOR region exhibits a complex organization when studied by various basespecific fluorochromes and silver staining. The DNA-specific fluorochromes are also useful tools in cytophotometric DNA measurements.     

When an amide is more like histidine than imidazole: the role of axial ligands in heme catalysis

 Of the many subtle protein-cofactor interactions which facilitate oxidative catalysis by heme enzymes, the role of the axial ligand has for some time appeared to be fairly well understood. Recent studies from several laboratories, however, have provided good reason to reemphasize the importance of secondary interactions between the axial ligand and protein, as the results suggest that simple ligand identity is neither necessary nor sufficient for function. It has been widely proposed that the strong hydrogen bond between a proximal carboxylate and the histidine ligand of peroxidases assists O–O bond heterolysis and stabilizes the Fe(IV)=O center that is produced. Recent replacements of the axial ligand in a number of heme proteins have produced a few surprises, suggesting that the subtle interactions between the ligand and protein may in some cases be more important than the actual identity of the ligand. Received and accepted: 7 May 1996     

Ligand binding processes in hemoglobin. Chemical reactivity of iron studied by XANES spectroscopy.

K-absorption edge of coordinated ions exhibits a fine structure (through the use of XANES, or x-ray absorption near edge structures) that reflects the electronic repartition and the chemical reactivity of these ions. Comparative analysis of iron K-absorption-edge shape for hemoglobin derivatives with different ligand affinity suggests strongly that in hemoglobin, iron-forms with high and low affinity are highly improbable.     

Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques

The dissociation of the subunits of human adult oxyhemoglobin has been investigated by using steady-state fluorescence anisotropy, multifrequency phase fluorometry, and high hydrostatic pressure. Human hemoglobin obtained by using two purification procedures (bulk preparation by centrifugation or further fractionation using anion-exchange chromatography) was labeled with an extrinsic fluorescent probe, 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl). The long fluorescence lifetime of this probe allows for the observation of the macromolecular tumbling, and thus provides a method for observing changes in the size of the complex upon subunit dissociation under differing solution conditions of proton and organic phosphate concentration. At pH 7, the dansylated preparations of bulk and fractionated hemoglobin showed a concentration-dependent decrease in the anisotropy which though not identical can only arise from the tetramer to dimer dissociation. We observed primarily the dimer at pH 9 and a small destabilization of the tetramer in the presence of saturating inositol hexaphosphate (IHP). High-pressure experiments allowed for the observation of the dissociation of the hemoglobin dimer into monomers. From these measurements, we estimate the dimer dissociation constant to be between 0.1 and 1 nM. We compare the present results on the subunit affinities in hemoglobin obtained from steady-state and time-resolved fluorescence data with those obtained previously by using gel filtration, sedimentation, and kinetic techniques. These comparisons are indicative of a certain degree of conformational heterogeneity in the hemoglobin preparations.