Biomedical subjects

B M Hoffman

Publications and source records attributed to B M Hoffman.

At least 19 recordsLinked to original sources

PubMed2001

Interaction of acetylene and cyanide with the resting state of nitrogenase alpha-96-substituted MoFe proteins.

The nitrogenase MoFe protein contains the active site metallocluster called FeMo-cofactor [7Fe-9S-Mo-homocitrate] that exhibits an S = 3/2 EPR signal in the resting state. No interaction with FeMo-cofactor is detected when either substrates or inhibitors are incubated with MoFe protein in the resting state. Rather, the detection of such interactions requires the incubation of the MoFe protein together with its obligate electron donor, called the Fe protein, and MgATP under turnover conditions. This indicates that a more reduced state of the MoFe protein is required to accommodate substrate or inhibitor interaction. In the present work, substitution of an arginine residue (alpha-96(Arg)) located next to the active site FeMo-cofactor in the MoFe protein by leucine, glutamine, alanine, or histidine is found to result in MoFe proteins that can interact with acetylene or cyanide in the as-isolated, resting state without the need for the Fe protein, or MgATP. The dithionite-reduced, resting states of the alpha-96(Leu)-, alpha-96(Gln)-, alpha-96(Ala)-, or alpha-96(His)-substituted MoFe proteins show an S = 3/2 EPR signal (g = 4.26, 3.67, 2.00) similar to that assigned to FeMo-cofactor in the wild-type MoFe protein. However, in contrast to the wild-type MoFe protein, the alpha-96-substituted MoFe proteins all exhibit changes in their EPR spectra upon incubation with acetylene or cyanide. The alpha-96(Leu)-substituted MoFe protein was representative of the other alpha-96-substituted MoFe proteins examined. The incubation of acetylene with the alpha-96(Leu) MoFe protein decreased the intensity of the normal FeMo-cofactor signal with the appearance of a new EPR signal having inflections at g = 4.50 and 3.50. Incubation of cyanide with the alpha-96(Leu) MoFe protein also decreased the FeMo-cofactor EPR signal with concomitant appearance of a new EPR signal having an inflection at g = 4.06. The acetylene- and cyanide-dependent EPR signals observed for the alpha-96(Leu)-substituted MoFe protein were found to follow Curie law 1/T dependence, consistent with a ground-state transition as observed for FeMo-cofactor. The microwave power dependence of the EPR signal intensity is shifted to higher power for the acetylene- and cyanide-dependent signals, consistent with a change in the relaxation properties of the spin system of FeMo-cofactor. Finally, the alpha-96(Leu)-substituted MoFe protein incubated with (13)C-labeled cyanide displays a (13)C ENDOR signal with an isotropic hyperfine coupling of 0.42 MHz in Q-band Mims pulsed ENDOR spectra. This indicates the existence of some spin density on the cyanide, and thus suggests that the new component of the cyanide-dependent EPR signals arise from the direct bonding of cyanide to the FeMo-cofactor. These data indicate that both acetylene and cyanide are able to interact with FeMo-cofactor contained within the alpha-96-substituted MoFe proteins in the resting state. These results support a model where effective interaction of substrates or inhibitors with FeMo-cofactor occurs as a consequence of both increased reactivity and accessibility of FeMo-cofactor under turnover conditions. We suggest that, for the wild-type MoFe protein, the alpha-96(Arg) side chain acts as a gatekeeper, moving during turnover in order to permit accessibility of acetylene or cyanide to a specific [4Fe-4S] face of FeMo-cofactor.

Acetylene↗

PubMed2001

High-frequency and field EPR investigation of (8,12-diethyl-2,3,7,13,17,18-hexamethylcorrolato)manganese(III).

High-field and frequency electron paramagnetic resonance (HFEPR) of solid (8,12-diethyl-2,3,7,13,17,18-hexamethylcorrolato)manganese(III), 1, shows that in the solid state it is well described as an S = 2 (high-spin) Mn(III) complex of a trianionic ligand, [Mn(III)C(3)(-)], just as Mn(III) porphyrins are described as [Mn(III)P(2)(-)](+). Comparison among the structural data and spin Hamiltonian parameters reported for 1, Mn(III) porphyrins, and a different Mn(III) corrole, [(tpfc)Mn(OPPh(3))], previously studied by HFEPR (Bendix, J.; Gray, H. B.; Golubkov, G.; Gross, Z. J. Chem. Soc., Chem. Commun. 2000, 1957-1958), shows that despite the molecular asymmetry of the corrole macrocycle, the electronic structure of the Mn(III) ion is roughly axial. However, in corroles, the S = 1 (intermediate-spin) state is much lower in energy than in porphyrins, regardless of axial ligand. HFEPR of 1 measured at 4.2 K in pyridine solution shows that the S = 2 [Mn(III)C(3)(-)] system is maintained, with slight changes in electronic parameters that are likely the consequence of axial pyridine ligand coordination. The present result is the first example of the detection by HFEPR of a Mn(III) complex in solution. Over a period of hours in pyridine solution at ambient temperature, however, the S = 2 Mn(III) spectrum gradually disappears leaving a signal with g = 2 and (55)Mn hyperfine splitting. Analysis of this signal, also observable by conventional EPR, leads to its assignment to a manganese species that could arise from decomposition of the original complex. The low-temperature S = 2 [Mn(III)C(3)(-)] state is in contrast to that at room temperature, which is described as a S = 1 system deriving from antiferromagnetic coupling between an S = (3/2) Mn(II) ion and a corrole-centered radical cation: [Mn(II)C(*)(2-)] (Licoccia, S.; Morgante, E.; Paolesse, R.; Polizio, F.; Senge, M. O.; Tondello, E.; Boschi, T. Inorg. Chem. 1997, 36, 1564-1570). This temperature-dependent valence state isomerization has been observed for other metallotetrapyrroles.

Journal Article↗

PubMed2001

Cryoreduction of methyl-coenzyme M reductase: EPR characterization of forms, MCR(ox1) and MCR (red1).

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methyl-coenzyme M (CH(3)S-CH(2)CH(2)SO(3)) from methane. The active site is a nickel tetrahydrocorphinoid cofactor, factor 430, which in inactive form contains EPR-silent Ni(II). Two such forms, denoted MCR(silent) and MCR(ox1)(-)(silent), were previously structurally characterized by X-ray crystallography. We describe here the cryoreduction of both of these MCR forms by gamma-irradiation at 77 K, which yields reduced protein maintaining the structure of the oxidized starting material. Cryoreduction of MCR(silent) yields an EPR signal that strongly resembles that of MCR(red1), the active form of MCR; and stepwise annealing to 260-270 K leads to formation of MCR(red1). Cryoreduction of MCR(ox1)(-)(silent) solutions shows that our preparative method for this state yields enzyme that contains two major forms. One behaves similarly to MCR(silent), as shown by the observation that both of these forms give essentially the same redlike EPR signals upon cryoreduction, both of which give MCR(red1) upon annealing. The other form is assigned to the crystallographically characterized MCR(ox1)(-)(silent) and directly gives MCR(ox1) upon cryoreduction. X-band spectra of these cryoreduced samples, and of conventionally prepared MCR(red1) and MCR(ox1), all show resolved hyperfine splitting from four equivalent nitrogen ligands with coupling constants in agreement with those determined in previous EPR studies and from (14)N ENDOR of MCR(red1) and MCR(ox1). These experiments have confirmed that all EPR-visible forms of MCR contain Ni(I) and for the first time generated in vitro the EPR-visible, enzymatically active MCR(red1) and the activate-able "ready" MCR(ox1) from "silent" precursors. Because the solution Ni(II) species we assign as MCR(ox1)(-)(silent) gives as its primary cryoreduction product the Ni(I) state MCR(ox1), previous crystallographic data on MCR(ox1)(-)(silent) allow us to identify the exogenous axial ligand in MCR(ox1) as the thiolate from CoM; the cryoreduction experiments further allow us to propose possible axial ligands in MCR(red1). The availability of model compounds for MCR(red1) and MCR(ox1) also is discussed.

Binding Sites↗

PubMed2001

Resonance Raman and EPR investigations of the D251N oxycytochrome P450cam/putidaredoxin complex.

We have performed resonance Raman and electron paramagnetic resonance (EPR) studies on the dioxygen bound state of the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putidaredoxin (Pd). The D251N oxy-P450cam/Pd complex has a perturbed proton delivery mechanism and shows a significantly red-shifted UV-visible spectrum as observed in Benson et al. [Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Biochemistry 36, 5104-5107]. The red shift has been interpreted to indicate a major perturbation of the electronic structure of the oxy-heme complex. However, we find no evidence that electron transfer has occurred from Pd to the heme active site of D251N oxy-P450cam. This suggests that both electron and proton transfer are perturbed by the D251N mutation and that these processes may be coupled. Three oxygen isotope sensitive Raman features are identified in the Pd complex, and occur at 1137, 536, and 399 cm(-1). These values are not significantly different from those for WT or D251N oxy-P450cam. However, a careful examination of the oxygen stretching feature near 1137 cm(-1) reveals the presence of three peaks at 1131, 1138, and 1146 cm(-1), which we attribute to the presence of conformational substates in oxy-P450cam. A significant change in the conformational substate population is observed for the D251N oxy-P450cam when the Pd complex is formed. We suggest that the conformational population redistribution of oxy-P450cam, along with the red-shifted electronic spectra, reflects a structural equilibrium of the oxy-heme that is perturbed upon Pd binding. We propose that this structural perturbation is connected to the effector function of Pd and may involve changes in the electron donation properties of the thiolate ligand.

Amino Acid Substitution↗

PubMed2001

Solitaire and gemini metallocene porphyrazines.

We report the synthesis and physical characterization of a series of peripherally functionalized porphyrazines (pzs) of the forms H2[pz(A;B3)] and trans-H2[pz(A2);B2], where A is a dithiolene chelate of molybdocene or vanadocene and B is a solublizing group. The precursor pz's 8 and 9, of the form H2[pz(A;B3)], where A = (4-(butyloxycarbonyl)-S-benzyl)2 and B = di-tert-butylphenyl (8) or di-n-propyl (9), have been prepared, deprotected, and peripherally metalated with molybdocene and vanadocene to form 1(Mo(IV)) and 1(V(IV)), prepared from 8, and 2(Mo(IV)) from 9, respectively. Likewise, the protected trans-H2[pz(A2);B2)], where A = (S-benzyl)2 and B = 3,6-butyloxybenzene (12) or A = (S-benzyl)2 and B = (tert-butylphenyl)2 (13), have been prepared and peripherally metalated with molybdocene and vanadocene to give the trans dinuclear complexes, 3(Mo(IV),Mo(IV)), 3(V(IV),V(IV)) (from 12), and 4(V(IV),V(IV)) (from 13). A crystal structure of the trans vanadocene pz 4(V(IV),V(IV)) is presented; the distance between the two vanadium atoms is 14.5 A. The molybdocene-appended pz's are highly redox active and exhibit cyclic voltammograms that are more than just the sum of the metallocene and the parent pz's. Chemical oxidation with FcPF6 gives the Mo(V) species 1(Mo(V)), 2(Mo(V)), 3(Mo(V),Mo(IV)), and 3(Mo(V),Mo(V)). Their EPR spectra are indicative of extensive delocalization from the Mo(V) into the dithiolato-pz. The EPR spectrum of the mononuclear paramagnetic vanadocene pz, 1(V(IV)), shows an expected 8-line pattern for an S = 2 system with hyperfine coupling to a single 51V (I = 7/2) nucleus, but the dinuclear vanadocene pz's, 3(V(IV),V(IV)) and 4(V(IV),V(IV)), exhibit a striking 15-line pattern of the same breadth from the S = 1 state formed by exchange coupling between the S = 2 vanadium centers of a dinuclear complex. Thus, the porphyrazine macrocycle is capable of mediating magnetic exchange interactions between metal ions bound to the periphery, separated by 14.5 A.

Journal Article↗

PubMed2001

35 GHz ENDOR characterization of the "very rapid" signal of xanthine oxidase reacted with 2-hydroxy-6-methylpurine (13C8): evidence against direct Mo-C8 interaction.

Xanthine oxidase is a molybdenum-containing enzyme that catalyzes the hydroxylation of xanthine and a wide variety of other aromatic heterocycles. In the course of the reaction with xanthine and substrates such as 2-hydroxy-6-methylpurine (HMP), the enzyme gives rise to a Mo(V) EPR signal, denoted "very rapid", that arises from an authentic catalytic intermediate. The two alternative catalytic mechanisms proposed for this enzyme differ critically in whether the distance between Mo and C8 of the purine nucleus in this intermediate is short enough to admit a direct bonding interaction. To examine this distance, we have performed 13C ENDOR measurements of the "very rapid" EPR signal generated by xanthine oxidase during reaction with 13C8-HMP. The resulting (13)C8 hyperfine tensor, A = [10.2(1), 7.0(1), 6.5(1)] MHz, is discussed in the framework of a detailed consideration of factors involved in extracting metrical parameters from an anisotropic hyperfine interaction composed of contributions from multiple sources, in particular, the effect of the local contributions from spin density on (13)C8. The analysis presented here gives a Mo...C distance whose value is expected to be ca. 2.7-2.9 A in the "very rapid" intermediates formed with both xanthine and HMP, consistent with plausible bond lengths for a Mo-O-C8 fragment where C8 is a trigonal-planar aromatic carbon. The difference from earlier conclusions is explained. The data thus do not support the existence of a direct Mo-C bond in the signal-giving species. This conclusion supports a mechanism that does not involve such an interaction and which begins with base-assisted nucleophilic attack of the Mo(VI)-OH group on the C-8 of substrate, with concomitant hydride transfer to the Mo=S group to give Mo(IV)-SH; the EPR-active "very rapid" species then forms by one-electron oxidation and deprotonation to yield the EPR-detectable Mo(V)OS(OR) species. We further discuss the complexities and limitations of the semiempirical method used to arrive at these conclusions.

Electron Spin Resonance Spectroscopy↗

PubMed2001

Optical, magnetic, and electronic properties of peripherally fused macrocycles: molybdocene porphyrazines.

Metal-free and copper porphyrazines, [H(2)pz] and [Cu pz], have been fused at the periphery with molybdocene dithiolene, [Cp(2)Mo]. The optical, magnetic, and electronic properties of the resulting neutral and cationic complexes are studied, using first-principles density functional theory implemented by the discrete variational method. Analysis of the charge and spin distribution shows that the porphyrazine core is strongly coupled with the peripheral complex. The calculated optical absorption is found to be in reasonable agreement with experimental spectra, lending support to our theoretical model. Under appropriate circumstances one observes interaction of unpaired spins localized in the vicinity of both metal sites. The calculated spin distribution shows that [Cp(2)Mo][Cu pz] and [Cp(2)Mo][H(2)pz](+) have a magnetic moment of 1 micro(B) while [Cp(2)Mo][Cu pz](+) and [Cp(2)Mo][H(2)pz] have no moment, in good agreement with the results of X-band EPR spectra. The Cu-Mo magnetic interaction is antiferromagnetic, being mediated by pyrrol nitrogens, meso nitrogens, carbons, and sulfurs.

Journal Article↗

PubMed2001

Hydroxylation of camphor by reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes.

We have employed gamma-irradiation at cryogenic temperatures (77 K and also approximately 6 K) of the ternary complexes of camphor, dioxygen, and ferro-cytochrome P450cam to inject the "second" electron of the catalytic process. We have used EPR and ENDOR spectroscopies to characterize the primary product of reduction as well as subsequent states created by annealing reduced oxyP450, both the WT enzyme and the D251N and T252A mutants, at progressively higher temperatures. (i) The primary product upon reduction of oxyP450 4 is the end-on, "H-bonded peroxo" intermediate 5A. (ii) This converts even at cryogenic temperatures to the hydroperoxo-ferriheme species, 5B, in a step that is sensitive to these mutations. Yields of 5B are as high as 40%. (iii) In WT and D251N P450s, brief annealing in a narrow temperature range around 200 K causes 5B to convert to a product state, 7A, in which the product 5-exo-hydroxycamphor is coordinated to the ferriheme in a nonequilibrium configuration. Chemical and EPR quantitations indicate the reaction pathway involving 5B yields 5-exo-hydroxycamphor quantitatively. Analogous (but less extensive) results are seen for the alternate substrate, adamantane. (iv) Although the T252A mutation does not interfere with the formation of 5B, the cryoreduced oxyT252A does not yield product, which suggests that 5B is a key intermediate at or near the branch-point that leads either to product formation or to nonproductive "uncoupling" and H(2)O(2) production. The D251N mutation appears to perturb multiple stages in the catalytic cycle. (v) There is no spectroscopic evidence for the buildup of a high-valence oxyferryl/porphyrin pi-cation radical intermediate, 6. However, ENDOR spectroscopy of 7A in H(2)O and D(2)O buffers shows that 7A contains hydroxycamphor, rather than water, bound to Fe(3+), and that the proton removed from the C(5) carbon of substrate during hydroxylation is trapped as the hydroxyl proton. This demonstrates that hydroxylation of substrates by P450cam in fact occurs by the formation and reaction of 6. (vi) Annealing at > or = 220 K converts the initial product state 7A to the equilibrium product state 7, with the transition occurring via a second nonequilibrium product state, 7B, in the D251N mutant; in states 7B and 7 the hydroxycamphor hydroxyl proton no longer is trapped. (vii) The present results are discussed in the context of other efforts to detect intermediates in the P450 catalytic cycle.

Camphor↗

PubMed2001

Determination of the hemoglobin surface domains that react with cytochrome b5.

We have compared the photoinitiated electron-transfer (ET) reaction between cytochrome b(5) (b(5)) and zinc mesoporphyrin-substituted hemoglobin [(ZnM)Hb] and Hb variants in order to determine whether b(5) binds to the subunit surface of either or both Hb chains, or to sites which span the dimer--dimer interface. Because the dimer--dimer interface would be disrupted for monomers or alpha beta dimers, we studied the reaction of b(5) with alpha ZnM chains and (ZnM)Hb beta W37E, which exists as alpha beta dimers in solution. Triplet quenching titrations of the ZnHb proteins with Fe(3+)b(5) show that the binding affinity and ET rate constants for the alpha-chains are the same when they are incorporated into a Hb tetramer or dimer, or exist as monomers. Likewise, the parameters for beta-chains in tetramers and dimers differ minimally. In parallel, we have modified the surface of the Hb chains by neutralizing the heme propionates through the preparation of zinc deuterioporphyrin dimethyl ester hemoglobin, (ZnD-DME)Hb. The charge neutralization increases the ET rate constants 100-fold for the alpha-chains and 40-fold for the beta-chains (but has has little effect on the affinity of either chain type for b(5), similar to earlier results for myoglobin). Together, these results indicate that b(5) binds to sites at the subunit surface of each chain rather than to sites which span the dimer-dimer interface. The charge-neutralization results further suggest that b(5) binds over a broad area of the subunit face, but reacts only in a minority population of binding geometries.

Animals↗

PubMed2001

Synthesis of near-IR absorbing/emitting porphyrazine derivatives with tunable solubility.

We report the synthesis of porphyrazines (pzs), or tetraazaporphyrins, of the form H2[pz(An;B4-n)], where A is [S(CH2)3COOR]2 (R = n-Pr,H) and B is a fused beta,beta'-diisopropyloxybenzo group, including the compounds with n = 4 (6), n = 3 (7) and the trans compound with n = 2 (8) (Scheme 1). The synthesis employs Linstead crossover macrocyclization of dimethyl 6,7-dicyano-5,8-dithia-6(Z)-dodecenedioate, MNT(C4O2Me)2 (2), with 1-imino-4,7-bis(1-methylethoxy)-1H-isoindole-3-amine (4). These pigments were characterized by 1H NMR, 13C NMR, absorbance/fluorescence spectroscopy, mass spectrometry, and microanalysis. An X-ray crystal structure of 8 is presented. Of particular note, 6-8 display intense near-IR absorbance and dual UV-visible/near-IR emission which are very important in potential biomedical applications, both for cancer therapy (photodynamic therapy, PDT) and cancer diagnosis (optical tumor imaging). For example, the trans-porphyrazine 8 has an intense long-wavelength absorption at ca. 800 nm (log epsilon = 4.18) and S1 fluorescence at approximately 820 nm, where mammalian tissue is effectively penetrated by light. Transformation of the ester group permits a wide range of functionality and solubility to be generated without change in optical properties. As an example, hydrolysis of these compounds by LiOH in THF/H2O gives the corresponding carboxylato-functionalized pigments 9-11, which are described. The last of these dissolves without aggregation in fetal calf serum.

Journal Article↗

PubMed2000

Calculation of z-coordinates and orientational restraints using a metal binding tag.

We introduce a new simple methodology allowing the measurement of (1)H-(15)N residual dipolar couplings, dipolar shifts, and unpaired electron-amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance. We show that this tag can be substituted with cobalt and manganese. Binding of cobalt to the gag zinc finger-barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field. Partial alignment permitted measurement of (1)J(HN) scalar couplings along with dipolar couplings. Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only (1)J(HN) scalar couplings. Dipolar couplings, ranging from -0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc. The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron-amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.

Bacterial Proteins↗

PubMed2000

Preparation of polyetherol-appended sulfur porphyrazines and investigations of peripheral metal ion binding in polar solvents.

We describe the complete synthesis and characterization of a new family of peripherally functionalized porphyrazines (pz's) with four, three, or two (in a trans conformation) bis[thioethoxy(ethoxy)ethanol] moieties appended at the pyrroles. These "polyetherol" groups serve as weak exocyclic binding sites for a number of metal ions and also provide solubility of the pz's in low molecular-weight alcohols and water. Electronic spectra of the modified porphyrazines exhibit distinct changes in the visible region (both absorbance and fluorescence) in response to treatment with Ag+, Pb2+, Cd2+, Cs+, and Ni2+ in solution. Such properties make these compounds intriguing candidates for incorporation into the transducer layers in optically based chemical sensors.

Ethers↗

PubMed2000

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis.

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.

Animals↗

PubMed2000

Binding of octa-plus porphyrazines to DNA.

We present a preliminary report of the interaction of octacationic N-methyl-pyridyl metalloporphyrazines, M=Cu(II) (Cupz+8) and Zn(II) (Znpz+8), with calf thymus DNA. These were monitored by electronic absorption spectroscopy, and in the case of Znpz+8, also by emission spectroscopy. These studies show that both the Cupz+8 and Znpz+8 interact strongly with DNA, and at sufficient concentrations induce a highly colored precipitate. The spectrum of Cupz+8 red-shifts as DNA is added, which is interpreted as indicative of pi stacking of the charged macrocycle as it binds electrostatically to the outside of the DNA duplex. The spectrum of Znpz+8 does not shift, indicative of simple electrostatic surface binding. The absence of stacking may be related to the presence of an axial metal-ion ligand for Znpz+8, presumably 1120, while the metal ion of Cupz+8 is four-coordinate.

Animals↗

PubMed2000

Bis(dimethylamino)porphyrazines: synthetic, structural, and spectroscopic investigations.

The synthesis and isolation of unsymmetrical porphyrazines bearing two, four, and six bis-(dimethylamino) functionalities has been achieved via the base-catalyzed cross-condensation of 1,2-dicyanobenzene 8 and bis(dimethylamino)maleonitrile 7. In addition, the benzo-fused hexaaminoporphyrazine dimer 10 was prepared from condensation of dinitrile 7 (in excess) with benzenebis(1,3-diiminopyrroline) 9. Electrochemical studies reveal that all porphyrazines may be readily oxidized. The X-ray structures of porphyrazines 2b and 5a and the cis isomer 3a are presented. The latter is the first structure of a porphyrazine having a cis-type substitution pattern. The extended pi-conjugation in dimer 10 causes a approximately 100 nm red-shifted Q-band in the electronic absorption spectrum.

Crystallography, X-Ray↗

PubMed2000

Extension of transverse relaxation-optimized spectroscopy techniques to allosteric proteins: CO- and paramagnetic fluoromet-hemoglobin [beta (15N-valine)].

We present the first steps in applying transverse relaxation-optimized spectroscopy (TROSY) techniques to the study of allosterism. Each beta-chain of the hemoglobin (Hb) tetramer has 17 valine residues. We have (15)N-labeled the beta-chain Val residues and detected 16 of the 17 (1)H-(15)N correlation peaks for beta-chain Val of the R state CO-Hb structure by using the TROSY technique. Sequence-specific assignments are suggested, based mainly on analysis of the (1)H pseudocontact-shift increments produced by oxidizing the diamagnetic R state HbCO to the paramagnetic R state fluoromet form. When possible, we support these assignments with sequential nuclear Overhauser effect (NOE) information obtained from a two-dimensional [(1)H,(1)H]-NOESY-TROSY experiment (NOESY, NOE spectroscopy). We have induced further the R-T conformational change by adding the allosteric effector, inositol hexaphosphate, to the fluoromet-Hb sample. This change induces substantial increments in the (1)H and (15)N chemical shifts, and we discuss the implication of these findings in the context of the tentative sequence assignments. These preliminary results suggest that amide nitrogen and amide proton chemical shifts in a selectively labeled sample are site-specific probes for monitoring the allosteric response of the ensemble-averaged solution structure of Hb. More important, the chemical-shift dispersion obtained is adequate to permit a complete assignment of the backbone (15)N/(13)C resonances upon nonselective labeling.

Allosteric Site↗

PubMed2000

Nitrogenase reduction of carbon disulfide: freeze-quench EPR and ENDOR evidence for three sequential intermediates with cluster-bound carbon moieties.

Freeze-quenching of nitrogenase during reduction of carbon disulfide (CS(2)) was previously shown to result in the appearance of a novel EPR signal (g = 2.21, 1.99, and 1.97) not previously associated with any of the oxidation states of the nitrogenase metal clusters. In the present work, freeze-quench X- and Q-band EPR and Q-band (13)C electron nuclear double resonance (ENDOR) spectroscopic studies of nitrogenase during CS(2) reduction disclose the sequential formation of three distinct intermediates with a carbon-containing fragment of CS(2) bound to a metal cluster inferred to be the molybdenum-iron cofactor. Modeling of the Q-band (35 GHz) EPR spectrum of freeze-trapped samples of nitrogenase during turnover with CS(2) allowed assignment of three signals designated "a" (g = 2.035, 1.982, 1.973), "b" (g = 2.111, 2.002, and 1.956), and "c" (g = 2.211, 1. 996, and 1.978). Freezing samples at varying times after initiation of the reaction reveals that signals "a", "b", and "c" appear and disappear in sequential order. Signal "a" reaches a maximal intensity at 25 s; signal "b" achieves maximal intensity at 60 s; and signal "c" shows maximal intensity at 100 s. To characterize the intermediates, (13)CS(2) was used as a substrate, and freeze-trapped turnover samples were examined by Q-band (13)C ENDOR spectroscopy. Each EPR signal ("a", "b", and "c") gave rise to a distinct (13)C signal, with hyperfine coupling constants of 4.9 MHz for (13)C(a), 1. 8 MHz for (13)C(b), and 2.7 MHz for (13)C(c). Models for the sequential formation of intermediates during nitrogenase reduction of CS(2) are discussed.

Azotobacter vinelandii↗

PubMed2000

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation.

We have studied four proteins containing oxidized 3Fe clusters ([Fe3S4]+, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T1, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2-4.2 K. The T1, values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g3 values and in 57 Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T1 is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at approximately 20 cm(-1) above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported 57Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J13=J, J12=J(1+epsilon'), J23=J(1+epsilon), where Jij is the isotropic exchange coupling between ferric ions i and j, and epsilon' and epsilon' are measures of coupling inequivalence. We have extended their theory to include the effects of epsilon' not equal to 0 and thus derived an exact expression for the energy of the doublet excited state for any epsilon, epsilon'. This excited state energy corresponds roughly to epsilonJ and is in the range 5-10 cm(-1) for each of these four 3Fe proteins. This magnitude of the product epsilonJ, determined by our time-domain relaxation studies in the temperature range 2-4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5-50 K, NMR studies in the range 293-303 K, and static susceptibility measurements in the range 1.8-200 K. We suggest that an apparent disagreement as to the individual values of J and epsilon be resolved in favor of the values obtained by susceptibility and NMR (J > or approximately 200 cm(-1) and epsilon> or =0.02 cm(-1)). as opposed to a smaller J and larger r as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters.

Aconitate Hydratase↗