The characterization and magnetic properties of the azide and imidazole derivatives of Pseudomonas nitrite reductase

Optical absorption, mcd, and epr spectroscopy have been used to characterize the azide and imidazole derivatives of oxidized Pseudomonas nitrite reductase. At pH 7.0 azide binds solely to heme d1 with an affinity constant, Kaff = 360 M-1, whereas imidazole binds to both hemes c and d1 with kaff = 35 and 55 M-1, respectively. Low-temperature mcd and epr spectroscopy indicate that c and d1 are low-spin ferrihemes in both derivatives, although the epr of the heme d1-azide component is very weak and requires explanation. Attempts to obtain a high-spin heme d1 in the intact enzyme using the weak field ligands fluoride and thiocyanate have proved unsuccessful. Electron paramagnetic resonance experiments involving an oxidized enzyme derivatives in which heme d1 is complexed by NO, and hence epr silent, have enabled unambiguous assignment of the epr spectrum of Pseudomonas nitrite reductase.     

Metal phthalocyanine substituted hemoglobins. Complexes of manganese and zinc tetrasulfonated phthalocyanines with apohemoglobin

Artificial hemoglobins have been prepared with Mn(III) and Zn(II) tetrasulfonated phthalocyanines in place of heme. Their structure and properties have been investigated by difference spectroscopy, CD, epr, electrophoresis, and molecular weight estimation.Spectrophotometric titration data indicate the ratio of the reagents in this process to be 1:1. The visible absorption spectra show the main peak at 625 nm for the manganese compound and 681 nm for the zinc one. It is evident from CD experiments that incorporation of Mn(III)L into apohemoglobin increases helical content of the protein whereas that of Zn(II)L increases its unfolding due to the change of electronic configuration of Zn(II) ion on coordination with the protein.On the basis of spectroscopic and epr data, the formula of the manganese complex is suggested to be (O)Mn(IV)L-globin, whereas that of the zinc complex Zn(II)L-globin. Electrophoresis and molecular weight estimation indicate both complexes to be dimers.Manganese complex binds additional ligands as CN^?, imidazole, CO, and NO. Spectroscopic and epr data indicate reduction of the manganese complex and formation of the NO adduct with probable formula (NO)⁺Mn(II)L-globin. Mechanism of this process is suggested.Both phthalocyanine globins are not able to combine reversibly with oxygen and cannot act as physiological oxygen carriers.     

Spectroscopic Properties of Siroheme Extracted from Sulfite Reductases

Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two pK values, 4.2 and 9.0, were determined by pH titration in the range 2–12. The first pK (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second pK (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with g_XXX = 6.0 and ^g= 2.0, typical of high-spin ferric hemes (S = 5/2), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with g_XXX = 2.38 and _g = 1.76 that is indicative of a low-spin ferric heme (S = 1/2). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

Electron paramagnetic resonance of nitrosylprotoheme dimethyl ester complexes with imidazole derivatives as model systems for nitrosylhemoproteins.

For the purpose of understanding the electron paramagnetic resonance (epr) spectral change of nitrosylhemoproteins under various conditions, the epr spectra for the model system have been analyzed. The model system consists of the nitrogen oxide complex of the iron(II) protoporphyrin IX dimethyl ester and various imidazole derivatives (three hindered and six unhindered imidazole derivatives). The results of the analysis indicate the existence of two molecular species in the model system, which differ in structure of the FeNO unit. These observations were compared with those for the nitrosylhemoproteins.     

Interactions of 4-nitroquinoline 1-oxide with deoxyribodinucleotides.

The interactions of 4-nitroquinoline 1-oxide (NQO), a potent mutagen and carcinogen, with several self- and non-self-complementary deoxydinucleotides were probed by using absorption spectra of the charge transfer bands and 1H and 13C NMR spectra. Absorption spectra were analyzed by using Benesi-Hildebrand-type equations to yield stoichiometries and equilibrium constants of complex formation. Non-self complementary dimers form weak l:1 complexes [dpTpG:NQO, K(25 degrees C) = 22 M-1] while self-complementary dimers form strong 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA with NQO gives a 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA, K(25 degrees C) = 8.6 X 10(3) M-2]. Analyses of the changes in 13C and 1H NMR chemical shifts with complex formation gave approximate orientations for the intercalation of NQO with self-complementary dimer minihelixes. In the (dpCpG)2:NQO and (dpGpC)2:NQO complexes, the NO2 group of NQO probably lies in the major grove and the NO2, NO containing NQO ring is stacked near the purine imidazole ring. In the (dpTpA)2:NQO and (dpApT)2NQO complexes, the NO2 seems to project into the minor grove and the NQO benzenoid ring is over the purine imidazole ring.     

Paramagnetic centers and acetyl-coenzyme A/CO exchange activity of carbon monoxide dehydrogenase from Methanothrix soehngenii.

Carbon monoxide (CO) dehydrogenase was purified, both aerobically and anaerobically, to apparent homogeneity from Methanothrix soehngenii. The enzyme contained 18 +/- 2 (n = 6) mol Fe/mol and 2.0 +/- 0.1 (n = 6) mol Ni/mol. Electron paramagnetic resonance (EPR) spectra of the aerobically purified CO dehydrogenase showed one sharp EPR signal at g = 2.014 with several characteristics of a [3Fe-4S]1+ cluster. The integrated intensity of this signal was low, 0.03 S = 1/2 spin/alpha beta dimer. The 3Fe spectrum was not affected by incubation with CO or acetyl-coenzyme A, but could be reduced by dithionite. The spectrum of the reduced, aerobically purified enzyme showed complex EPR spectra, which had several properties typical of two [4Fe-4S]1+ clusters, whose S = 1/2 spins weakly interacted by dipolar coupling. The integrated intensity was 0.1-0.2 spin/alpha beta dimer. The anaerobically isolated enzyme showed EPR spectra different from the reduced aerobically purified enzyme. Two major signals were apparent. One with g values of 2.05, 1.93 and 1.865, and an Em7.5 of -410 mV, which quantified to 0.9 S = 1/2 spin/alpha beta dimer. The other signal with g values of 1.997, 1.886 and 1.725, and an Em7.5 of -230 mV gave 0.1 spin/alpha beta dimer. When the enzyme was incubated with its physiological substrate acetyl-coenzyme A, these two major signals disappeared. Incubation of the enzyme under CO atmosphere resulted in a partial disappearance of the spectral component with g = 1.997, 1.886, 1.725. Acetyl-coenzyme A/CO exchange activity, 35 nmol.min-1.mg-1 protein, which corresponded to 7 mol CO exchanged min-1 mol-1 enzyme, could be detected in anaerobic enzyme preparations, but was absent in aerobic preparations. Carbon dioxide also exchanged with C-1 of acetyl-coenzyme A, but at a much lower rate than CO and to a much lower extent.     

Paramagnetic centers of carbon monoxide dehydrogenase from aceticlastic Methanosarcina barkeri

Carbon monoxide dehydrogenase from Methanosarcina barkeri, purified to 95% homogeneity, contains 30 Fe, 2 Ni, 1 Zn, and 1 Cu (per alpha 2 beta 2 enzyme). Core extrusion experiments indicate 6 [4Fe-4S] clusters/tetramer, and electron paramagnetic resonance (epr) spectroscopy detects at least one of these clusters, in the reduced form, with apparent g values of 2.05, 1.94, and 1.90, and Em9.2-390 mV. A second epr signal, also seen in the reduced enzyme, has apparent g values of 2.005, 1.91, and 1.76, and Em9.2-35 mV. Two signals were seen in thionin-oxidized enzyme, one with a line shape suggestive of Cu(II), and the other resembling that of a [3Fe-4S] cluster. The enzymes nonphysiological substrate, CO, caused several spectral changes to the reduced enzyme, most notably a shift of the g = 1.76 feature to g = 1.73.     

Reactions of molybdenum-sulphur compounds with cyanide: chemical evolution and deactivation of molybdoenzymes.

Reactions of molybdenum-sulphur compounds with cyanide are reported which may be relevant to (1) the chemical evolution of molybdoenzymes and (2) deactivation of molybdoenzymes by cyanide. (1) With aqueous cyanide MoS2 gave thio-bridged complex anions [(Mo(CN)6)2(mu-S)]6- and [(Mo(CN)4(mu-S))2]6-. Under prebiotic conditions such complexes could have been formed similarly from molybdenite and may have been precursors of molybdoenzymes. (2) Only those compounds which contained terminal sulphur bound to molybdenum (i.e., Mo = S groups), viz. oxothiomolybdates and the complex [(Mo(mu-S)(S)(Et2NCS2))2], reacted with cyanide; thiocyanate was formed and the molybdenum underwent two-electron reduction. That the cyanolysable sulphur of xanthine oxidase reacts in the same way with cyanide suggests the presence of a Mo = S group which could be a structural feature of the enzyme or could have been formed by initial cyanolysis of a bound persulphide or cysteine residue.     

Manganese cytochrome c. Structure and properties.

Oxidized and reduced manganese cytochromes c, Mn Cyt c+ and Mn Cyt c, have been synthesized. Mn Cyt c+ and Fe Cyt c+ have identical electrophoretic and ion exchange mobilities. Mn Cyt c+ does not bind F-, CN-, or N3- ions; Mn Cyt c does not bind CO or O2. Mn Cyt c is very rapidly autooxidized by O2 even at -50 degrees. The manganese ion is readily dissociated from Mn Cyt c at acidic pH values. Both Mn Cyt c and Mn Cyt c+ are high spin complexes with 3d5 S = 5/2 and 3d4 S = 2 electronic configurations, respectively. The epr spectrum of Mn Cyt c is rhombic with (formula: see text). Both oxidized and reduced Mn Cyt c react with NO; the former reaction is reversible and the product has the following epr spectral parameters: (formula: see text). There is no superhyperfine interaction observable with the NO ligand, and the unpaired electron density is estimated to be mostly in the metal ion d xy orbital. The structure is best formulated as Mn Cyt c (NO)+. The half-reduction potential of Mn Cyt c is + 60 +/- 40 mV. It is neither oxidized by cytochrome oxidase nor reduced by NADH, NADPH, or succinate cytochrome reductase. These physical, chemical, and enzymic properties of manganese cytochromes c suggest a five-coordinate metalloporphyrin prosthetic group with the manganese ion situated significantly out-of-plane toward the side of His-18.     

CO dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in CO2 and argon atmospheres

The EPR and redox properties of the metal complexes in CO dehydrogenase (CODH) from Clostridium thermoaceticum were studied. Controlled potential coulometric reductive titrations of CODH were performed under argon and CO2 atmospheres. In the titrations performed under argon, five to eight electrons/dimer were required for reduction, and four distinct EPR signals appeared. These included a signal with gave = 1.82 (Em approximately -220 mV), two signals with the same g values but different linewidths at gave = 1.94 (Em approximately -440 mV), and a signal at gave = 1.86 (Em approximately -530 mV). All of the S = 1/2 EPR signals had low spin concentrations; values between 0.2 and 0.3 spins/dimer were typically obtained for each signal. Features between g = 6 and 4, typical of S = 3/2 states, were also observed, and these may account, at least to some degree, for the low spin concentration values. Under CO2, and at negative potentials, CODH served as an electrocatalyst in the reduction of CO2 to CO. The apparent half-maximal activity for this reduction at pH 6.3 occurred at -430 mV, a potential near the thermodynamic value. An EPR signal, arising from a complex containing Ni, Fe, and the carbon from CO/CO2 developed along with this activity. The reduction of this complex is probably the last step to occur prior to the catalysis of CO2 reduction.     

1H-NMR and rosonance Raman spectra of octaethylporphyrinatoiron(III) perchlorate and its mono imidazole adduct

The 1H-NMR spectra and the resonance Raman spectra of intermediate spin complex, octaethylporphyrinatoiron (III) perchlorate (OEP-Fe(III)ClO4) and its mono imidazole adduct have been recorded and analyzed. The perchlorate complex was determined to be an intermediate-spin state (S = 3/2) in dichloromethane. The mono imidazole and 2-methylimidazole adducts of OEP-Fe(III)ClO4 were of the high-spin state in dichloromethane, which is a good model for the ferrihemoproteins such as metmyoglobins. The spin state of OEP-Fe(III)ClO4 varies the polarity of solvent from typical high-spin (S = 5/2) to typical low-spin (S = 1/2) state including intermediate-spin state (S = 3/2). The resonance Raman studies of the intermediate-spin complex in various solvents indicate that the complex is a plausible model to reproduce anomalous physico-chemical properties of the ferricytochrome c' at physiological condition.     

[Nitrotyrosyl]cytochrome c. Studies of the effect of iron binding, protein denaturants and oxidation-reduction potentials.

Static measurements of the reaction of ligand binding were done by conventional spectrophotometry. The ligand-binding reactions with nitrated cytochrome c were performed with imidazole, iminazole, CO and NO. The stoicheiometry was found to be 1:1, and the stability constants for the complexes formed between the nitrated cytochrome c and the ligands are: 2.58 X 10(4) M-1 (imidazole); 1.01 X 10(2) M-1 (iminazole); 3.6 X 10(4) M-1 (CO); 2.74 X 10(4) M-1 (NO). It was found that the electrometric potentials at pH 7.0 and 25degreesC of [aminotyrosyl]cytochrome c are E'o form II = 0.115 V and E'o form I = 0.260 V, where forms I and II are two species of protein co-existing in the protein solution. The isoelectric point for the oxidized form of [nitrotyrosyl]cytochrome c was 10.05, at 4degreesC.     

The reaction of cytochrome oxidase with cyanide. Preparation of the rapidly reacting form and its conversion to the slowly reacting form

The magnitude of the slow phase of reaction of cytochrome oxidase with cyanide has been correlated with the size of the epr signal at g' = 12. This epr signal was not found in submitochondrial particles, and significant g' = 12 epr was only observed late in the purification of solubilized enzyme. The Hartzell-Beinert procedure for the purification of cytochrome oxidase (Hartzell, C.R., and Beinert, H. (1974) Biochim. Biophys. Acta 368, 318-338) has been modified so that the purified enzyme reacts in a single rapid phase with potassium cyanide and lacks the g' = 12 epr signal. This enzyme could be converted to the slowly reacting form upon incubation at low pH and/or low enzyme concentration. No procedure for the stable reversal of the process could be found. Some physical and chemical properties of the two forms of the enzyme are compared.     

Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.     

Spectral studies of human erythrocyte catalase.

The optical absorption and circular dichroic spectra of human erythrocyte catalase (EC 1.11.1.6) and its cyanide, azide, and fluoride derivatives over the wavelength range of 210 to 700 nm are reported. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the heme group from the protein. The fractions of the protein structure present as alpha helix, beta pleated sheet, and unordered structure have been estimated from the CD spectrum in the far-ultraviolet region. The CD spectra also indicate that the protein conformation does not change appreciably after cyanide binding. The epr spectroscopy of the native enzyme and its cyanide complex are reported. The spectral results are compared with catalase obtained from other mammalian and bacterial sources.     

Spectroscopic studies on heme d in the visible and infrared

Heme d has been isolated from the terminal oxidase complex of Escherichia coli strain MR43L/F152 and purified by high-pressure liquid chromatography. The infrared spectrum indicated that carbonyls in the chlorin skeleton of this isolated heme existed as carboxylic acids. Earlier work on the iron-free chlorin had demonstrated the presence of a spirolactone substituent. This may have arisen from a cyclization reaction from a dicarboxylic acid, diol precursor. Although the free heme in extracts can exist as a diol, this does not prove that the diol as such is the precise form in the enzyme complex. Visible and fluorescence spectra are reported for a variety of derivatives and complexes of heme d to establish a spectral library that may be used to prove the presence of this structure in other enzymes or cells. Association constants have been measured for complexes of heme d with cyanide, imidazole, and pyridine and are contrasted to available data for protoheme.     

Probing subtle coordination changes in the iron-quinone complex of photosystem II during charge separation,by the use of NO

The terminal electron acceptor of Photosystem II, PSII, is a linear complex consisting of a primary quinone, a non-heme iron(II), and a secondary quinone, Q(A)Fe(2+)Q(B). The complex is a sensitive site of PSII, where electron transfer is modulated by environmental factors and notably by bicarbonate. Earlier studies showed that NO and other small molecules (CN(-), F(-), carboxylate anions) bind reversibly on the non-heme iron in competition with bicarbonate. In the present study, we report on an unusual new mode of transient binding of NO, which is favored in the light-reduced state (Q(A)(-)Fe(2+)Q(B)) of the complex. The related observations are summarized as follows: (i) Incubation with NO at -30 degrees C, following light-induced charge separation, results in the evolution of a new EPR signal at g = 2.016. The signal correlates with the reduced state Q(A)(-)Fe(2+) of the iron-quinone complex. (ii) Cyanide, at low concentrations, converts the signal to a more rhombic form with g values at 2.027 (peak) and 1.976 (valley), while at high concentrations it inhibits formation of the signals. (iii) Electron spin-echo envelope modulation (ESEEM) experiments show the existence of two protein (14)N nuclei coupled to electron spin. These two nitrogens have been detected consistently in the environment of the semiquinone Q(A)(-) in a number of PSII preparations. (iv) NO does not directly contribute to the signals, as indicated by the absence of a detectable isotopic effect ((15)NO vs (14)NO) in cw EPR. (v) A third signal with g values (2.05, 2.03, 2.01) identical to those of an Fe(NO)(2)(imidazole) synthetic complex develops slowly in the dark, or faster following illumination. (vi) In comparison with the untreated Q(A)(-)Fe(2+) complex, the present signals not only are confined to a narrow spectral region but also saturate at low microwave power. At 11 K the g = 2.016 signal saturates with a P(1/2) of 110 microW and the g = 2.027/1.976 signal with a P(1/2) of 10 microW. (vii) The spectral shape and spin concentration of these signals is successfully reproduced, assuming a weak magnetic interaction (J values in the range 0.025-0.05 cm(-)(1)) between an iron-NO complex with total spin of (1)/(2) and the spin, (1)/(2), of the semiquinone, Q(A)(-). The different modes of binding of NO to the non-heme iron are examined in the context of a molecular model. An important aspect of the model is a trans influence of Q(A) reduction on the bicarbonate ligation to the iron, transmitted via H-bonding of Q(A) with an imidazole ligand to the iron.     

Proton NMR study of coordinated imidazoles in low-spin ferric heme complexes. Assignment of single proton histidine resonance in hemoproteins.

The proton signals for the coordinated axial imidazoles in a series of low-spin ferric bis-imidazole complexes with natural porphyrin derivatives have been located and assigned. The methyl signals of several methyl-substituted imidazoles have also been resolved for the mixed ligand complexes of imidazole and cyanide ion. The imidazole spectra for the bis complexes are essentially the same as those reported earlier for synthetic porphyrins, with the hyperfine shifts exhibiting comparable contributions from the dipolar and contact interactions. The contact contribution reflects spin transfer into a vacant imidazole pi orbital. The spectra of both the mono- and bis-imidazole complex concur in predicting that only the 2-H and 5-CH2 signals of an axial histidine are likely to resonate clearly outside the diamagnetic 0 to --10 ppm from TMS region in hemoproteins. However, both the 2-H and 4-H imidazole peaks are found to be too broad to detect in a hemoprotein. Hence, it is suggested that the pair of non-heme, single-proton resonances in low-spin met-myoglobin cyanides arise from the non-equivalent methylene protons at the 5-position of the histidyl imidazole. Both the resonance positions and relative linewidths in the model compounds are consistent with the data for this pair of protons in myoglobins. The possible interpretations of the average downfield bias of these signals as well as the magnitude of their spacing, are discussed in terms of the conformation of the proximal histidine relative to the heme group.