Hypothyroidism in patients treated with radiotherapy for head and neck carcinoma: standardised long-term follow-up study.

OBJECTIVE: Hypothyroidism is a common complication when radiotherapy is part of the treatment for head and neck tumours. This study aimed to show the incidence of hypothyroidism and possible risk factors in these patients. METHODS: Factors related to the population, tumour, treatment and occurrence of hypothyroidism were analysed in 241 patients diagnosed with head and neck carcinoma. RESULTS: Approximately 53 per cent of patients were diagnosed with radiation-induced hypothyroidism. Its occurrence was related to: tumour location, laryngeal surgery type, neck dissection type, post-operative complications, cervical radiotherapy and radiotherapy unit type (linear particle accelerator or telecobalt therapy technology). CONCLUSION: Control of thyroid function should be standardised for several years after treatment, particularly in patients with risk factors, such as those treated with telecobalt therapy, those with post-operative complications and for whom the thyroid parenchyma is included in the irradiated area (laryngeal or pharyngeal location and bilateral cervical radiation).

Effects of mirtazapine on the sleep wake rhythm of geriatric patients with major depression: an exploratory study with actigraphy.

INTRODUCTION: Major depression and insomnia are among the most frequent neuropsychiatric syndromes in the geriatric population. Although most SSRI antidepressants affect sleep continuity, mirtazapine has been found to improve sleep continuity in patients with depression. The aim of the present study was to assess by actigraphic recordings changes in sleep patterns of geriatric patients with major depression before and during treatment with mirtazapine (30 mg). METHODS: Patients aged 60 years or more with major depressive disorder were recruited at the outpatient service of a specialized mental health centre. Severity of depression was rated with the Montgomery-Asberg depression rating scale and subjective perception of sleep was assessed with the Pittsburgh sleep quality index (PSQI). Actigraphic parameters were registered 4 days before the onset of mirtazapine treatment (patients were drug free in this period of time) and recorded at day 60 of treatment with mirtazapine. RESULTS: A significant decrease was observed in the sleep fragmentation index. While a significant improvement was observed in the subjective assessment of quality after treatment with mirtazapine, actigraphic measures of sleep parameters did not show changes in line with mirtazapine treatment. DISCUSSION: Mirtazapine produces minimal changes on actigraphic measures in the sleep of elderly outpatients. Sleep produced by mirtazapine indicates a more pronounced effect in ≥ 80-year-old patients. This differential response should be considered during treatment of this clinical population.

Hipoglucemia por hiperinsulinemia endógena en el adulto / Hypoglycemia due to endogenous hyperinsulinism in an adult

Introducción: La nesidioblastosis es una causa conocida de hipoglucemia en recién nacidos. Sin embargo, en adultos permanece poco entendida, creando un dilema tanto diagnóstico como terapéutico. Exponemos el caso de un paciente varón de 63 años que presenta hipoglucemia hiperinsulinémica en el que no se identificó la existencia de insulinoma antes ni durante la cirugía, practicándosele una pancreatectomía subtotal. La histología mostró la existencia de una nesidioblastosis. Conclusión: Identificamos un paciente con hipoglucemia hiperinsulinémica grave ocasionada por una proliferación difusa de los islotes pancreáticos, por lo que pensamos que esta entidad debe ser tenida en consideración ante un paciente que se intervenga con el diagnóstico de insulinoma no identificado previamente. En estos casos debe considerarse la realización de una pancreatectomía subtotal, a pesar de que el tratamiento óptimo está por determinar

Introduction: Nesidioblastosis is a well-known cause of hypoglycemia in neonates. However, it is poorly understood in adults, creating both diagnostic and therapeutic dilemmas. Case report: A 63-year-old man presented with hyperinsulinemic hypoglycemia. An insulinoma was not identified prior to or during surgery, and subtotal pancreatectomy was performed. Histological examination revealed the presence of nesidioblastosis. Conclusion: We identified a patient with severe hyperinsulinemic hypoglycemia due to diffuse islet cell disease. This etiology should always be considered in patients with a preoperative presumptive diagnosis of insulinoma. In these cases, partial pancreatectomy is indicated, although the optimal treatment remains to be determined

Hipotiroidismo primario posradioterapia en pacientes con cáncer de cabeza y cuello / Primary hypothyroidism after radiotherapy in patients with head and neck cancer

Introducción: El tratamiento con radioterapia en el cáncer de cabeza y cuello es una causa de hipotiroidismo. Nos proponemos estudiar el desarrollo de hipofunción tiroidea en estos pacientes. Pacientes y métodos: Se realizó un análisis retrospectivo de 194 pacientes tratados con radioterapia por cáncer de cabeza y cuello. Se determinó la tirotropina (TSH) y la tiroxina (T4) libre a los 3, los 6 y los 12 meses después de la radioterapia y anualmente durante el seguimiento. El hipotiroidismo se clasificó como subclínico (aumento de la TSH y la T4 libre normal) y clínico (aumento de la TSH y disminución de la T4 libre). Se analizó la relación del hipotiroidismo con edad, sexo, estirpe histológica del tumor, dosis de radiación y el uso de quimioterapia. Resultados: Con una media de seguimiento de 4,2 años, 56 pacientes presentaron elevación de la TSH (39 pacientes subclínico y 17 clínico). El tiempo medio para el diagnóstico fue de 3,0 ± 1,8 años (subclínico 2,6 ± 1,5 años, clínico 4 ± 1,9 años; p < 0,05). El 80% de los pacientes fueron diagnosticados entre el segundo y el sexto año desde la radioterapia. La edad, la histología del tumor, la dosis de radiación y el uso de quimioterapia no modificaron la probabilidad de desarrollar hipotiroidismo. El sexo demostró un valor predictor (un 66,6 frente a un 25,8% en mujeres y varones, respectivamente; p < 0,05). Conclusiones: La tasa de incidencia de hipotiroidismo después de radioterapia en pacientes con cáncer de cabeza y cuello es elevada. Es importante determinar la TSH durante un largo período de tiempo después de la radioterapia (AU)

Introduction: Radiotherapy in the treatment of cancer of the head and neck can cause hypothyroidism. Our aim was to study the development of thyroid hypofunction in these patients. Patients and methods: We performed a retrospective analysis of 194 patients who underwent radiotherapy for head and neck cancer. Thyroid-stimulating hormone (TSH) and free thyroxine (FT4) levels were determined at 3, 6 and 12 months after radiotherapy and then annually during follow-up. Hypothyroidism was classified as subclinical (SHT) (an increase of TSG and normal FT4 levels) and clinical (CHT) (an increase of TSH and a decrease of FT4). The association between hypothyroidism and age, sex, histological type of the tumor, radiation dose and use of chemotherapy was analyzed. Results: With a mean follow-up of 4.2 years, 56 patients showed elevated TSH levels (SHT in 39 patients and CHT in 17). The mean time to diagnosis was 3.0 ± 1.8 years (SHT 2.6 ± 1.5 years, CHT 4 ± 1.9 years; p < 0.05). Eighty percent of the patients were diagnosed between the second and the sixth year after radiotherapy. Age, histological type, radiation dose and use of chemotherapy did not affect the probability of developing hypothyroidism. Sex had a predictive value (66.6% in women versus 25.8% in men; p < 0.05). Conclusions: The incidence rate of hypothyroidism after radiotherapy in patients with cancer of the head and neck is high. Prolonged follow-up of TSH levels should be performed in these patients after radiotherapy (AU)

Plasma lipids and blood fluidity in patients with polygenic hypercholesterolaemia treated with fluvastatin.

The clinical benefit brought about by HMG-CoA reductase inhibitors (statins) may not entirely be due to their lipid-lowering effect. Further investigation is necessary in order to determine the significance of ancillary effects to the clinical benefit of statin treatment. We studied 27 polygenic hypercholesterolaemia (PHC) patients before and 3 and 6 months after fluvastatin treatment. A control group of 38 normal, sex and age matched, subjects were also studied. The following parameters were measured: haematimetry, serum lipids and general biochemistry, apo-A/B and lipoproteins, fibrinogen, blood filterability, red blood cell aggregation, blood and plasma viscosity. PHC patients showed lower blood filterability (16.00+/-0.99 vs 19.90+/-2.90 microl/s), higher plasma fibrinogen (274.8+/-41.5 vs 241.6+/-43.2 mg/dl), increased erythrocyte aggregation at low shear stress (8.10+/-1.15 vs 7.19+/-1.29) and increased plasma viscosity (1.26+/-0.06 vs 1.23+/-0.05 mPa.s). Notable lipid changes after 6 months fluvastatin treatment were not accompanied by measurable changes in the haemorheological alterations of the PHC patients.

Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes.

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.

Shared control of hepatic glycogen synthesis by glycogen synthase and glucokinase.

We have used recombinant adenoviruses (AdCMV-RLGS and AdCMV-GK) to overexpress the liver isoforms of glycogen synthase (GS) and glucokinase (GK) in primary cultured rat hepatocytes. Glucose activated overexpressed GS in a dose-dependent manner and caused the accumulation of larger amounts of glycogen in the AdCMV-RLGS-treated hepatocytes. The concentration of intermediate metabolites of the glycogenic pathway, such as glucose 6-phosphate (Glc-6-P) and UDP-glucose, were not significantly altered. GK overexpression also conferred on the hepatocyte an enhanced capacity to synthesize glycogen in response to glucose, as described previously [Seoane, Gómez-Foix, O'Doherty, Gómez-Ara, Newgard and Guinovart (1996) J. Biol. Chem. 271, 23756-23760], although, in this case, they accumulated Glc-6-P. When GS and GK were simultaneously overexpressed, the accumulation of glycogen was enhanced in comparison with cells overexpressing either GS or GK. Our results are consistent with the hypothesis that liver GS catalyses the rate-limiting step of hepatic glycogen synthesis. However, hepatic glycogen deposition from glucose is submitted to a system of shared control in which the 'controller', GS, is, in turn, controlled by GK. This control is indirectly exerted through Glc-6-P, which 'switches on' GS dephosphorylation and activation.

Identification of two essential glutamic acid residues in glycogen synthase.

The detailed catalytic mechanism by which glycosyltransferases catalyze the transfer of a glycosyl residue from a donor sugar to an acceptor is not known. Through the multiple alignment of all known eukaryotic glycogen synthases we have found an invariant 17-amino acid stretch enclosed within the most conserved region of the members of this family. This peptide includes an E-X(7)-E motif, which is highly conserved in four families of retaining glycosyltransferases. Site-directed mutagenesis was performed in human muscle glycogen synthase to analyze the roles of the two conserved Glu residues (Glu-510 and Glu-518) of the motif. Proteins were transiently expressed in COS-1 cells as fusions to green fluorescence protein. The E510A and E518A mutant proteins retained the ability to translocate from the nucleus to the cytosol in response to glucose and to bind to intracellular glycogen. Although the E518A variant had approximately 6% of the catalytic activity shown by the green fluorescence protein-human muscle glycogen synthase fusion protein, the E510A mutation inactivated the enzyme. These results led us to conclude that the E-X(7)-E motif is part of the active site of eukaryotic glycogen synthases and that both conserved Glu residues are involved in catalysis. We propose that Glu-510 may function as the nucleophile and Glu-518 as the general acid/base catalyst.

Intracellular distribution of hepatic glucokinase and glucokinase regulatory protein during the fasted to refed transition in rats.

We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1- and 2-h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.

Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase.

Glucokinase (GK), a key enzyme in the glucose homeostatic responses of the liver, changes its intracellular localisation depending on the metabolic status of the cell. Rat liver GK and Xenopus laevis GK, fused to the green fluorescent protein (GFP), concentrated in the nucleus of cultured rat hepatocytes at low glucose and translocated to the cytoplasm at high glucose. Three mutant forms of Xenopus GK with reduced affinity for GK regulatory protein (GKRP) did not concentrate in the hepatocyte nuclei, even at low glucose. In COS-1 and HeLa cells, a blue fluorescent protein (BFP)-tagged version of rat liver GK was only able to accumulate in the nucleus when it was co-expressed with GKRP-GFP. At low glucose, both proteins concentrated in the nuclear compartment and at high glucose, BFP-GK translocated to the cytosol while GKRP-GFP remained in the nucleus. These findings indicate that the presence of and binding to GKRP are necessary and sufficient for the proper intracellular localisation of GK and directly involve GKRP in the control of the GK subcellular distribution.

Glycogenin, the primer of glycogen synthesis, binds to actin.

We have studied the intracellular localization of glycogenin by fusing green fluorescent protein (GFP) to the N-terminus of rabbit muscle glycogenin and expressing the chimeric protein in C2C12, COS-1 and rat hepatic cells. The fusion protein showed a nuclear and cytosolic distribution and partially co-localized with actin in the cytosol. Disruption of the actin cytoskeleton with cytochalasin D led to a change in the pattern of green fluorescence, which coincided with that observed for the remaining non-depolymerized actin. The distribution of the single point mutant K324A was completely uniform and was not affected by this drug. These findings indicate that rabbit muscle glycogenin binds to actin through the heptapeptide 321DNIKKKL327, a common motif found in other actin-binding proteins, which is located at the C-terminal end of this protein, and suggest that the actin cytoskeleton plays an important role in glycogen metabolism.

Muscle glycogen synthase translocates from the cell nucleus to the cystosol in response to glucose.

We have studied the intracellular localization of muscular glycogen synthase by fusing the green fluorescent protein (GFP) of the jelly-fish Aequorea victoria to the N-terminus of human muscle glycogen synthase (HMGS), and expressing the chimeric protein in C2C12, COS-1 cells, and primary cultured rat hepatocytes. In contrast to what we have recently found for the hepatic glycogen synthase (Fernandez-Novell et al. (1997) Biochem. J. 321, 227-231), the GFP/HMGS fusion protein is localized to the nucleus of the cell in the absence of glucose, and in the presence of the sugar it is essentially found in the cytosol. Insulin is not required for the translocation of the enzyme.

Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli.

A bond between the N delta of the imidazole ring of His 392 and the C beta of the essential Tyr 415 has been found in the refined crystal structure at 1.9 A resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the C beta atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392-Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80 degrees relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self-catalyzed heme conversion reaction and the formation of the His-Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound 1 is proposed.

Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant.

A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH-linked spectroscopic species. The electronic absorption and 1H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Soc. 114, 6592-6593; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1993) Biochemistry 32, 9807-9818]. Stopped-flow studies failed to detect even transient formation of a porphyrin-centered radical following addition of hydrogen peroxide to the Fe(III)-enzyme. The consequences of this drastic electrostatic modification of the active site on the steady-state kinetics of the variant are relatively minor.

Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant.

The structural and functional consequences of the introduction of a negatively charged amino acid into the active site of horse heart myoglobin have been investigated by replacement of the proximal Ser92 residue (F7) with an aspartyl residue (Ser92Asp). UV-visible absorption maxima of various ferrous and ferric derivatives and low-temperature EPR spectra of the metaquo (metMb) derivative indicate that the active site coordination geometry has not been perturbed significantly in the variant. 1H-NMR spectroscopy provides direct evidence for the existence of a distal water molecule as the sixth ligand in the oxidized form of the variant at pD 5.7. Spectrophotometric pH titration of the Ser92Asp variant is consistent with this finding and with a pKa = 8.90 +/- 0.02 [25.0 degrees C, mu = 0.10 M (NaCl)] for titration of the distal water molecule, identical to the value reported for the wild-type protein. X-ray crystallography of the metMb derivative indicates that the heme substituents conserve their orientations in the variant protein, except for a slight reorientation of the pyrrole A propionate group to which Ser92 normally hydrogen bonds and reorientation of the carboxyl end of the pyrrole D propionate group. No change is observed in conformation of the proximal (His93) or distal (Wat156) heme ligands. 1H-NMR spectroscopy of the metMbCN form of the protein indicates that a slight rotation of the proximal His93 ligand has occurred in this derivative. Resonance Raman experiments indicate increased conformational heterogeneity in the proximal pocket of the variant. Failure to detect electron density for the Asp residue in the X-ray diffraction map of the variant protein and high average thermal factors for the pyrrole A propionate substituent are consistent with this observation. The variant exhibits novel pH-dependent behavior in the metMb form, as shown by 1H-NMR spectroscopy, and provides evidence for a heme-linked titratable group with a pKa of 5.4 in this derivative. The metMbCN and deoxyMb derivatives also exhibit pH-dependent behavior, with pKas of 5.60 +/- 0.07 and 6.60 +/- 0.07, respectively, compared to the wild-type values of 5.4 +/- 0.04 and 5.8 +/- 0.1. The heme-linked ionizable group is proposed to be His97 in all three derivatives. The reduction potential of the variant is 72 +/- 2 mV vs SHE [25.0 degrees C, mu = 0.10 M (phosphate), pH 6.0], an increase of 8 mV over the wild-type value. The possible influence of a number of variables on the magnitude of the reduction potential in myoglobin and other heme proteins is discussed.

Structure of the heme d of Penicillium vitale and Escherichia coli catalases.

A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

pH, electrolyte, and substrate-linked variation in active site structure of the Trp51Ala variant of cytochrome c peroxidase.

Electronic absorption, MCD, and 1H NMR spectroscopy have been used to characterize the structures and linkage relationships of three active site states, LS1, HS, and LS2, of the Trp51Ala variant of yeast cytochrome c peroxidase (CcP) in the Fe(III) state. In addition, the binding of three substrates (styrene, catechol, and guaiacol) to the Fe(III) variant has been studied by 1H NMR spectroscopy, and the paramagnetically shifted resonances of the cyanide adduct of the variant have been assigned. The heme iron is hexacoordinated in all three pH-dependent states of the enzyme. LS1, the dominant acidic species, exhibits electronic and MCD spectra indicative of low-spin, bis-histidine coordination environment for the heme iron. The HS form, which dominates at intermediate pH, exhibits electronic, MCD, and 1H NMR spectra characteristic of high-spin heme Fe(III) with axial histidyl and water ligands. The LS2 species exhibits spectroscopic properties indicative of a bis-histidine, low-spin Fe(III) derivative. The equilibrium constants for interconversion of these forms of the variant enzyme are highly dependent on ionic strength, specific anions, and temperature of the solution, with the HS form stabilized relative to the other forms in the presence of several noncoordinating, anionic species. Aromatic substrates such as styrene, catechol, and guaiacol affect the chemical shifts of the heme substituents of the HS species but not of the LS2 species. Based on these results, a model is proposed that accounts to a large extent for the electrostatic origin of the three forms of the active site of the Trp51Ala variant and the mechanisms by which they are differentially stabilized in solution.

Trans effects on cysteine ligation in the proximal His93Cys variant of horse heart myoglobin.

Three variants of horse heart myoglobin (Mb) in which the proximal His93 residue has been replaced with a Cys residue have been constructed and studied by NMR, EPR, and MCD spectroscopy to evaluate the contributions of proximal and distal residues to the coordination environment of the heme iron in these proteins. Although no experimental conditions were identified that allowed quantitative ligation of the cysteine residue to the heme iron in the His93Cys variant, all of the spectroscopic evidence collected for the His93Cys/His64Ile and His93Cys/His64Val double variants supports the assignment of thiolate as the ligand to iron in the oxidized forms of these variants. The double metMb variants exhibit Soret maxima that are considerably blue-shifted, 1H NMR spectra with decreased mean methyl resonances, and EPR spectra with highly rhombic g values. These spectroscopic data for the Fe(III) variants resemble the corresponding properties reported for ferricytochrome P-450. The decrease in the reduction potential of the double variants by 280 mV relative to wild-type protein is also consistent with the low midpoint potential of cytochrome P-450. MCD spectroscopy of these variants confirms that the proximal cysteine residue is not bound in the reduced forms of these proteins and, in the case of the His93Cys variant, that the distal histidine is coordinated to the iron. Similar coordination environments were created in the ferrimyoglobin variants by cyanogen bromide modification, which resulted in cyanation of the sulfur atom and prevented the ligation of Cys93 to the heme iron.(ABSTRACT TRUNCATED AT 250 WORDS)

The proximal ligand variant His93Tyr of horse heart myoglobin.

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)