RESUMEN
The mammalian skeleton performs a diverse range of vital functions, requiring mechanisms of regeneration that restore functional skeletal cell populations after injury. We hypothesized that the Wnt pathway specifies distinct functional subsets of skeletal cell types, and that lineage tracing of Wnt-responding cells (WRCs) using the Axin2 gene in mice identifies a population of long-lived skeletal cells on the periosteum of long bone. Ablation of these WRCs disrupts healing after injury, and three-dimensional finite element modeling of the regenerate delineates their essential role in functional bone regeneration. These progenitor cells in the periosteum are activated upon injury and give rise to both cartilage and bone. Indeed, our findings suggest that WRCs may serve as a therapeutic target in the setting of impaired skeletal regeneration.
Asunto(s)
Proteína Axina/metabolismo , Huesos/citología , Huesos/lesiones , Regeneración Hepática , Animales , Proteína Axina/genética , Linaje de la Célula , Proliferación Celular , Análisis de Elementos Finitos , Ratones , Modelos Teóricos , Vía de Señalización WntRESUMEN
To understand the nature of the thyroid hormone binding site, we characterized the binding of 3,3',5-triiodo-L-thyronine (T3) and its analogues to eight naturally occurring mutated human beta 1 thyroid hormone receptors (h-TR beta 1). The mutant receptors were derived from patients with the syndrome of generalized thyroid hormone resistance, and each has a point mutation in the hormone binding domain (KT, R338W; TP, L450H; IR, D322H; NN, G347E; AH, P453H; OK, M442V; RL, F459C; and ED, A317T). Compared to the wild-type h-TR beta 1, binding of T3 was reduced by as much as 97% for the mutants. The order of binding affinity of wild-type h-TR beta 1 to the analogues is T3 > D-T3 > L-thyroxine > 3,5-diiodo-L-thyronine > 3,3',5'-triiodo-L-thyronine. The mutant receptors showed essentially the same order of reduced affinities for the analogues, but the amounts of the reductions varied in each case. These results suggest specific local interactions (interplay) of analogues with the mutated residues in the receptors. On the basis of these data and a putative structure of the hormone binding domains as an eight-stranded alpha/beta barrel, we propose the location of the hormone in the binding site of h-TR beta 1. Ionic bonds anchor the hormone's alanine side chain to loop 4 of the 8-fold alpha/beta barrel. The phenyl ring lies across the amino-terminal face of the domain with the phenoxy ring pointing downward into the barrel interacting with beta-strand 8 on the opposite side.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Mutación Puntual , Receptores de Hormona Tiroidea/metabolismo , Triyodotironina/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli , Humanos , Modelos Biológicos , Conformación Proteica , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triyodotironina/análogos & derivadosRESUMEN
Generalized resistance to thyroid hormone is an inherited disease characterized by unresponsiveness of pituitary and peripheral tissues to thyroid hormone. Genetic analysis of several kindreds linked this syndrome to the gene for the beta-form of the thyroid hormone receptor, and this led to the subsequent identification of various mutations in the ligand-binding domain of this receptor. In this region we now have found 4 new point mutations with reduced T3-binding affinities from separate kindreds by direct sequencing of polymerase chain reaction products. Similar to previously studied kindreds, the reduction in T3 binding of these four kindreds ranged from 2.5- to 5-fold, indicating that these are not neutral polymorphisms. Furthermore, the pattern of inheritance of these 4 kindreds is familial in 2, sporadic in 1, and unknown in 1. To date, 20 distinct mutations have been identified, of which 18 are clustered in 2 distinct topographical regions: 11 are within the tau i/dimerization subdomains of exon 9, and 7 are within the L2 subdomain of exon 10. The 4 newly identified mutations coupled to the 9 mutations our laboratory has previously identified provide new insights into the clinical aspects of generalized resistance to thyroid hormone. Kindreds with mutations in exon 9 compared with those in exon 10 have significantly more problems in language development, as manifested by articulation problems and/or wide discrepancies in verbal and performance IQs. Interestingly, marked variability in language deficiency as well as other clinical patterns were seen not only between kindreds but also within a kindred. Further identification and clinical correlations of new mutations will continue to enhance our understanding of the structure/function relationships and physiological role of the human thyroid hormone receptor.
Asunto(s)
Trastornos del Lenguaje/genética , Mutación/genética , Receptores de Hormona Tiroidea/genética , Adolescente , Adulto , Anciano , Secuencia de Bases , Niño , Preescolar , Exones/genética , Femenino , Humanos , Trastornos del Lenguaje/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Pruebas Neuropsicológicas , Fenotipo , Reacción en Cadena de la Polimerasa , Pruebas de Función de la TiroidesRESUMEN
The gene (pcaB) for 3-carboxymuconate lactonizing enzyme (CMLE; 3-carboxymuconate cycloisomerase; EC 5.5.1.2) from Pseudomonas putida has been cloned into pMG27NS, a temperature-sensitive expression vector, and expressed in Escherichia coli N4830. The specific activity and kinetic parameters of the recombinant CMLE were comparable to those previously reported. A comparison of the deduced amino acid sequence of CMLE with sequences available in the PIR and Genbank databases revealed that CMLE has highly significant sequence homology to the class II fumarase family, particularly to adenylosuccinate lyase from Bacillus subtilis. CMLE has no significant homology to muconate lactonizing enzyme (MLE) from P. putida, its sister enzyme in the beta-ketoadipate pathway. These findings fully corroborate a prediction made by us on the basis of mechanistic and stereochemical analyses of CMLE and MLE [Chari, R. V. J., Whitman, C. P., Kozarich, J. W., Ngai, K.-L., & Ornston, L. N. (1987) J. Am. Chem. Soc. 109, 5514-5519] and suggest that CMLE and MLE were recruited into this specialized pathway from two different enzyme families.
Asunto(s)
Fumarato Hidratasa/genética , Pseudomonas putida/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , Clonación Molecular , ADN Bacteriano , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Cinética , Datos de Secuencia Molecular , Plásmidos , Pseudomonas putida/genética , Homología de Secuencia de AminoácidoRESUMEN
Mutations in the gene encoding the human beta 1 T3 receptor (hTR beta 1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR beta 1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T3 affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T3 affinity required 5- and 15-fold higher T3 concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTR beta 1s were able to inhibit the function of transfected normal hTR beta 1 and endogenous retinoic acid receptor in activating a palindromic positive T3 response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T3 concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTR beta 1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTR beta 1 was assessed by immunocytochemistry. The hTR beta 1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells. These data suggest that different mutations in the ligand-binding domain of the human hTR beta 1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T3 receptor dimers.
Asunto(s)
Receptores de Hormona Tiroidea/metabolismo , Enfermedades de la Tiroides/metabolismo , Transcripción Genética , Triyodotironina/metabolismo , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Receptores de Hormona Tiroidea/genética , Enfermedades de la Tiroides/genética , TransfecciónRESUMEN
The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Racemasas y Epimerasas/metabolismo , Secuencia de Bases , Bromuros/metabolismo , Catálisis , Dicroismo Circular , Análisis Mutacional de ADN , Deuterio/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lisina/química , Datos de Secuencia Molecular , Oligonucleótidos/química , Racemasas y Epimerasas/genética , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Liasas Intramoleculares , Ácidos Mandélicos/metabolismo , Pseudomonas/genética , Acetolactato Sintasa/genética , Oxidorreductasas de Alcohol/genética , Aldehído Oxidorreductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Carboxiliasas/genética , Escherichia coli/metabolismo , Isomerasas/genética , L-Lactato Deshidrogenasa/genética , Datos de Secuencia Molecular , Operón , Pseudomonas/metabolismo , Piruvato Descarboxilasa/genética , Piruvato Oxidasa/genética , Racemasas y Epimerasas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Marcadores de Afinidad/metabolismo , Clonación Molecular , Genes Bacterianos , Genes , Isomerasas/genética , Ácidos Mandélicos/metabolismo , Mutación , Fenilpropionatos/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas/genética , Racemasas y Epimerasas/genética , Farmacorresistencia Microbiana/genética , Fenilpropionatos/farmacología , Pseudomonas/enzimología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/crecimiento & desarrollo , Racemasas y Epimerasas/aislamiento & purificación , Racemasas y Epimerasas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The recent development of a high-yield expression system and purification scheme for mandelate racemase has enabled us to produce sufficiently large quantities of pure enzyme to pursue x-ray crystallographic study. Large, single crystals of mandelate racemase have been grown from buffered polyethylene glycol (pH 8.0) in the presence of 10 mM magnesium chloride. The crystals grow in several habits, and we have identified two distinct tetragonal space groups in preliminary x-ray diffraction analysis. Crystals shaped as rectangular plates demonstrate 4/mmm Laue symmetry and systematic absences consistent with the space group I422. They have cell dimensions of a = b = 153 A and c = 181 A. Octahedrally shaped crystals of mandelate racemase display 4/m Laue symmetry and systematic absences consistent with the space group 14. Cell dimensions for these crystals are a = b = 113 A and c = 124 A. Based on estimates of Vm and on the measured density of the 1422 form, we suggest that two subunits of mandelate racemase (38,570 daltons/subunit) occupy the asymmetric unit in both crystal forms. Crystals of both forms diffract to beyond 3.0-A resolution. We are currently screening for isomorphous heavy-atom derivatives.
Asunto(s)
Isomerasas , Racemasas y Epimerasas , Conformación Proteica , Pseudomonas aeruginosa/enzimología , Difracción de Rayos XRESUMEN
The gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) was cloned in Pseudomonas aeruginosa (ATCC 15692). The selection for the cloned gene was based upon the inability of P. aeruginosa to grow on (R)-mandelate as sole carbon source by virtue of the absence of mandelate racemase in its mandelate pathway. Fragments of P. putida DNA obtained by digestion of chromosomal DNA with Sau3A were ligated into the BamHI site of the Gram-negative vector pKT230 and transformed into the P. aeruginosa host. A transformant able to utilize (R)-mandelate as sole carbon source was characterized, and the plasmid was found to contain approximately five kilobase pairs of P. putida DNA. Subcloning of this DNA revealed the position of the gene for the racemase within the cloned DNA from P. putida. The dideoxy-DNA sequencing procedure was used to determine the sequence of the gene and its translated sequence. The amino acid sequence and molecular weight for mandelate racemase deduced from the gene sequence (38 570) are in excellent agreement with amino acid composition and molecular weight data for the polypeptide recently determined with enzyme isolated from P. putida; these recent determinations of the polypeptide molecular weight differ significantly from the originally reported value of 69,500 [Fee, Judith A., Hegeman, G.D., & Kenyon, G.L. (1974) Biochemistry 13,2528], which was used to demonstrate that alpha-phenylglycidate, an active site directed irreversible inhibitor, binds to the enzyme with a stoichiometry of 1:1.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Genes , Isomerasas/genética , Pseudomonas/genética , Racemasas y Epimerasas/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Enzimas de Restricción del ADN , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Pseudomonas/enzimologíaRESUMEN
The interaction of Escherichia coli glutamine synthetase with beta, gamma-Cr(III)(H2O)4ATP (CrATP) has been studied. This substitution inert nucleotide functioned as an active site directed irreversible inhibitor of glutamine synthetase in solutions containing 15 mM MgCl2, 100 mM KCl, and 10 mM Pipes (pH 6.6). The inactivation reaction followed pseudo-first order saturation kinetics which demonstrated reversible binding of CrATP prior to the formation of inactive enzyme. CrATP was shown to be a competitive inhibitor versus MgATP. Also, significant protection was afforded by MgATP indicating that CrATP inactivates at the active site. Partial protection was afforded by glutamate or inorganic phosphate while inactivation was enhanced by Mn(II). The stoichiometry of CrATP incorporation was approximately one molecule per enzyme subunit, determined spectrophotometrically. Both the delta and lambda isomers of CrATP bound to glutamine synthetase, but only the lambda isomer was an active site directed irreversible inhibitor.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Marcadores de Afinidad , Cromo , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Sitios de Unión , Unión Competitiva , Escherichia coli/enzimología , Isomerismo , Cinética , MetalesRESUMEN
The purpose of the EPR and NMR studies presented in this article was to determine the spatial relationship between the n1 and n2 metal ion sites of E. coli glutamine synthetase. Table I presents the distances between these two metal ion sites in various complexes using Mn(II) bound to each site. These studies also employed the transition-state analog, methionine sulfoximine, as an active-site probe as well as various nucleotide complexes. Two primary conclusions result from these data. The Mn(II)-Mn(II) distance changes from approximately 10 to approximately 8 A when nucleotides bind to the enzyme presumably as the result of a protein conformational change. Two Mn(II) ions can bind to the enzyme in the presence of the substitution-inert Co(III)-ATP complex, implying that the metal ion [Co(III)] coordinated to the beta-gamma phosphoryl groups in the complex is displaced from the normal n2 metal ion site. A model showing the probable spatial relationships among components of the active site is shown in Fig. 6. This model comprises our current working hypothesis of the active site of glutamine synthetase. Further studies of distance relationships are presently underway in our laboratory and will be placed in the context of this model and the known kinetic mechanism.
Asunto(s)
Escherichia coli/enzimología , Glutamato-Amoníaco Ligasa/metabolismo , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón/métodos , Manganeso/análisis , Unión ProteicaRESUMEN
The distance between the two catalytically important metal ions of glutamine synthetase was determined by electron paramagnetic resonance (EPR). Mn(II) binds more tightly to the n1 site of this enzyme in the presence of methionine sulfoximine and the influence of Mn(II) bound at the n2 site on the EPR spectrum of Mn(II) at n1 was studied. A monotonic increase in the EPR spectrum of Mn(II) was observed at Mn:E (subunit) ratios of 0 to 0.8. After this point as Mn(II) was added to about 1.8 Mn:E, a decrease in the EPR signal was observed. This phenomenon was found for both adenylylated and unadenylylated forms of glutamine synthetase. The data were analyzed using a theory for dipolar electron-electron relaxation and a distance of 10-12 A was computed for the Mn(II)-Mn(II) separation. These data demonstrate that both modified and unmodified forms of glutamine synthetase which have different catalytic activities have a similar spatial relationship between the two catalytic metal ion sites.
Asunto(s)
Adenosina Monofosfato/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Manganeso/metabolismo , Sitios de Unión , Fenómenos Químicos , Química , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/enzimología , Matemática , Unión ProteicaRESUMEN
Yeast inorganic pyrophosphatase was found to bind two Mn2+ per subunit in the absence of phosphate and three Mn2+ per subunit in the presence of phosphate. Kinetic studies of the pyrophosphatase-catalyzed hydrolysis of Cr(NH3)4PP and Cr(H2O)4PP were carried out with Mn2+ and with Mg2+ as activators. The results from these studies suggest that three divalent cations per pyrophosphatase active site are required for catalysis. NMR and EPR studies were conducted to evaluate the relative location of the metal ion binding sites on the enzyme. The two Mn2+ ions bound to the free enzyme are in close enough proximity to magnetically interact. Analysis of the NMR and EPR data in terms of a dipolar relaxation mechanism between Mn2+ ions provides an estimate of the distance between them of 10-14 A. When the diamagnetic substrate analog [Co(NH3)4PNP]- or intermediate analog [Co(NH3)4 (P)2]- are bound to pyrophosphatase, two Mn2+ ions still bind to the enzyme and their magnetic interaction increases. In the presence of these Co3+ complexes, the Mn2+--Mn2+ separation decreases to 7-9 A. Several NMR and EPR experiments were conducted at low Mn2+ to pyrophosphatase ratios (approximately 0.3), where only one Mn2+ ion binds per subunit, in the presence of Cr3+ or Co3+ complexes of PNP or PP. Analysis of the Mn2+--Cr3+ dipolar relaxation evident in proton NMR and EPR data provided for the calculation of Mn2+--Cr3+ distances. When the substrate analog CrPNP was present, the Mn2+--Cr3+ distance was congruent to 7 A whereas, when Cr(P)2 was bound to pyrophosphatase, the Mn2+--Cr3+ distance was congruent to 5 A. These results strongly support a model for the catalytic site of pyrophosphatase that involves three metal ion cofactors.
Asunto(s)
Magnesio/farmacología , Manganeso/farmacología , Pirofosfatasas/metabolismo , Saccharomyces cerevisiae/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Pirofosfatasa Inorgánica , Cinética , Matemática , Unión ProteicaRESUMEN
The interaction of solvent water protons with the bound paramagnetic metal ions of beef heart cytochrome c oxidase has been examined. The observed proton relaxation rates of enzyme solutions had a negative temperature dependence, indicating a rapid exchange between solvent protons in the coordination sphere of the metal ions and bulk solvent. An analysis of the dependence of the proton relaxation rate on the observation frequency indicated that the correlation time, which modulates the interaction between solvent protons and the unpaired electrons on the metal ions, is due to the electron spin relaxation time of the heme irons of cytochrome c oxidase. This means that at least one of the hemes is exposed to solvent. The proton relaxation rate of the oxidized enzyme was found to be sensitive to changes in ionic strength and to changes in the spin states of the metal ions. Heme a3 was found to be relatively inaccessible to bulk solvent. Partial reduction of the enzyme caused a slight increase in the relaxation rate, which may be due to a change in the antiferromagnetic coupling between two of the bound paramagnetic centers. Further reduction resulted in a decreased relaxation rate, and the fully reduced enzyme was no longer sensitive to changes in ionic strength. The binding of cytochrome c to cytochrome c oxidase had little effect on the proton relaxation rates of oxidized cytochrome oxidase indicating that cytochrome c binding has little effect on solvent accessibility to the metal ion sites.