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Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew at a 40% slower rate than the wild type; however, it produced ethanol at a 31% higher rate. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicates that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy, including the upregulation of allantoin, urea, and amino acid uptake and utilization pathways and selective autophagic machinery. In addition, pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. The induction of a nitrogen starvation-like state and increase in lipid production in nitrogen-rich conditions suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and other yeasts for the production of lipids and lipid-derived compounds. • Deletion of URE2 increases ethanol and lipid production in Saccharomyces cerevisiae. • Deletion of URE2 reduces glycogen and trehalose production. • Metabolic changes mimic nitrogen starvation and autophagic response.

North-trending rifts and associated strike-slip faults in the Tibetan Plateau suggest Ceno zoic east-west extension, but the domi-nant modes of distributed extensional defor-ma tion and basin formation are unclear. The Lunggar basin in west-central Tibet is bounded by a

The human population and economy have grown exponentially and now have impacts on climate, ecosystem processes, and biodiversity far exceeding those of any other species. Like all organisms, humans are subject to natural laws and are limited by energy and other resources. In this article, we use a macro ecological approach to integrate perspectives of physics, ecology, and economics with an analysis of extensive global data to show how energy imposes fundamental constraints on economic growth and development. We demonstrate a positive scaling relationship between per capita energy use and per capita gross domestic product (GDP) both across nations and within nations over time. Other indicators of socioeconomic status and ecological impactare correlated with energy use and GDP. We estimate global energy consumption for alternative future scenarios of population growth and standards of living. Large amounts of energy will be required to fuel economic growth, increase standards of living, and lift developing...

Understanding the storage, flux, and turnover of nutrients in organisms is important for quantifying contributions of biota to biogeochemical cycles. Here we present a model that predicts the storage of phosphorus-rich RNA and whole-body phosphorus content in eukaryotes based on the mass- and temperature-dependence of ATP production in mitochondria. Data from a broad assortment of eukaryotes support the model's two main predictions. First, whole-body RNA concentration is proportional to mitochondrial density and consequently scales with body mass to the -1/4 power. Second, whole-body phosphorus content declines with increasing body mass in eukaryotic unicells but approaches a relatively constant value in large multicellular animals because the fraction of phosphorus in RNA decreases relative to the fraction in other pools. Extension of the model shows that differences in the flux of RNA-associated phosphorus are due to the size dependencies of metabolic rate and RNA concentration. Thus, the model explicitly links two biological currencies at the individual level: energy in the form of ATP and materials in the form of phosphorus, both of which are critical to the functioning of ecosystems. The model provides a framework for linking attributes of individuals to the storage and flux of phosphorus in ecosystems.

Summary 1. Recent studies have resurrected the debate over the value for the allometric scaling exponent that relates whole-organism metabolic rate to body size. Is it 3 / 4 or 2 / 3 ? This question has been raised before and resolved in favour of 3 / 4 . Like previous ones, recent claims for a value of 2 / 3 are based almost entirely on basal metabolic rate (BMR) in mammals. 2. Here we compile and analyse a new, larger data set for mammalian BMR. We show that interspecific variation in BMR, as well as field metabolic rates of mammals, and basal or standard metabolic rates for many other organisms, including vertebrates, invertebrates, protists and plants, all scale with exponents whose confidence intervals include 3 / 4 and exclude 2 / 3 . Our analysis of maximal metabolic rate gives a slope that is greater than and confidence intervals that exclude both 3 / 4 and 2 / 3 . 3. Additionally, numerous other physiological rates that are closely tied to metabolism in a wide variety of organisms, including heart and respiratory rates in mammals, scale as M − 1/4 . 4. The fact that quarter-power allometric scaling is so pervasive in biology suggests that different allometric relations have a common, mechanistic origin and provides an empirical basis for theoretical models that derive these scaling exponents.

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B). Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB+ on the pathway to and from the high-spin heme. The presence of CO on CuB+ suggests that O2 binding may be compromised in CO flow-flash experiments. Time-resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme (1 × 108 M−1 s−1) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t1/2 = 1.5 μs) of CO from CuB+. In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 109 to 1 × 108 M−1 s−1), but is still considerably faster (~10 μs at 1 atm O2) than the CO off-rate from CuB in the absence of O2 (milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB+ impedes the binding of O2 to CuB+ or, if O2 does not bind to CuB+ prior to heme a3, whether the CuB+-CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB+ in a concerted manner through steric and/or electronic effects. This would allow CuB+ to function as an electron donor during the fast (5 μs) breaking of the O–O bond. These results suggest that the binding of CO to CuB+ on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational Spectroscopies in Molecular Bioenergetics.

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) (DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982). The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in

The resonant Raman active mode identified in numerous studies as the heme iron−histidine stretch has been systematically investigated in the Raman spectrum of 15 exogenous ligands to the heme iron in the myoglobin proximal cavity mutant H93G. Mutation of the native histidine 93 of myoglobin to glycine (H93G) creates a cavity at the heme iron that can be filled with exogenous ligands. Substituted imidazoles and pyridines were introduced into the cavity at the heme iron of the deoxy ferrous H93G myoglobin by dialysis. Raman bands attributed to in-plane modes of the heme are unaffected by the change in heme−iron ligation. However, the Raman active band in the 180−250 cm-1 region ascribed to an iron−ligand out-of-plane mode is highly dependent on the identity of the axial ligand. Information on the normal mode was obtained using isotopically labeled imidazole, pyridine, and 1-methyl and 2-methyl imidazole. Relatively small isotope effects are observed for the heme−iron ligands imidazole H93G(Im) and pyridine ...

Notomastus lobatus chloroperoxidase (NCPO) and Amphitrite ornata dehaloperoxidase (DHP) catalyze the halogenation of phenols and dehalogenation of halophenols, respectively. Both enzymes require peroxide for activity and have recently been shown to contain histidine (His) as their proximal heme iron ligand. DHP is the only heme enzyme known to catalyze peroxide-dependent defluorination reactions, and NCPO is the first His-ligated heme-containing peroxidase capable of chlorinating halogen acceptors using chloride as the halogen donor. To probe the roles of the proximal His ligands in the mechanism of action of these two novel peroxidases, they have been examined by resonance Raman and infrared spectroscopy to determine the vibrational properties of their deoxyferrous and ferrous−CO derivatives. Previous studies of His-ligated heme proteins have revealed characteristic distinctions between dioxygen-binding globins and peroxidases. In the latter, the spectral properties are indicative of strong hydrogen bond...

The rotational barrier for MoW(meso-monotolyl octaethylporphyrin)2 (((TOEP)MoW(TOEP))) has been determined (¢G q rot ) 10.6 ( 0.1 kcal/mol) by variable-temperature NMR and complete band shape analysis and is compared with values previously obtained for the analogous homodimers. The overall quadruple bond strengths of these isostructural dimolybdenum, ditungsten, and molybdenum-tungsten porphyrin dimers have also been compared by calculation of the force constants corresponding to each metal-metal bond stretching frequency as observed by resonance Raman spectroscopy. The Raman results are as follows: (Mo(OEP))2, OMoMo ) 310 cm -1 , k ) 2.72 mdyn/A; ((OEP)MoW(OEP)),OMoW ) 279 cm -1 , k ) 2.89 mdyn/A; (Mo- (TOEP))2, OMoMo ) 310 cm -1 , k ) 2.72 OMoMo (W(TOEP))2, OWW ) 275 cm -1 , k ) 4.08 mdyn/A; and ((TOEP)- MoW(TOEP)), OMoW ) 278 cm -1 , k ) 2.87 mdyn/A. Both the 1 H NMR and Raman specta are consistent with a ((Por)MoW(Por)) structure wherein the Mo(Por) congener experiences a more drastic "bending-back" distortion of the porphyrin macrocycle.

The cyanide isotope-sensitive low-frequency vibrations of ferrous cyano complexes of cytochrome a3 are studied for cytochrome ba3 from Thermus thermophilus and cytochrome aa3 from bovine heart. Cyanide complexes of ba3 display three isotope sensitive frequencies at 512, 485, and 473 cm-1. The first is primarily an Fe-C stretching motion, whereas the lower wavenumber modes are bending motions. These iron-cyanide vibrations are independent of the redox levels of the other metal centers in the protein. On the other hand, the fully reduced bovine derivative complexed with cyanide gives rise to a bending vibration at 503 cm-1 and a stretching vibration at 469 cm-1. That is, the ordering of the stretching and bending frequencies is reversed from that of the bacterial protein. These results are analyzed by normal coordinate calculations to obtain comparative models for the binuclear O2 reducing site of the two proteins. We find that the observed frequencies are consistent with a linear Fe-C-N group and larger Fe-C stretching force constant (2.558 mdyn/A) for ba3 and a slightly bent Fe-C-N group (angle approximately 170 degrees) and a smaller Fe-C stretching force constant (2.335 mdyn/A) for aa3. Thus, there are significant differences in the interaction of cyanide with ferrous a3 in the two proteins that are most likely caused by a weaker proximal histidine interaction and stronger peripheral heme electron withdrawing effects in ba3. Possible sources of these protein-induced effects are discussed. Using the analysis developed here, comparison of the FeCN stretching and bending frequencies of the ferrous bovine a3-CN complex to those obtained from the ferric a3-CN complex suggests that upon conversion of the resting to the fully reduced protein, a conformational change occurs that constrains the ligand binding site.

Events following the photodissociation of the dinuclear rhodium(II,H) complex Rh 2 L 10 (L= CH 3 CN), are reported. Spectroscopic and electrochemical evidence demonstrates that this complex possesses novel reversible photochemistry with metastable photoproducts that exist for kiloseconds after photodissociation, and ultimately regenerate the original dimer. UV/Vis irradiation of acetonitrile solutions of the complex results in homolytic cleavage of the Rh Rh bond to give a promptly formed paramagnetic transient that EPR evidence suggests to be Rh(H)L 6 . This species has a half-life of ca. 50 s, and initiates a reaction sequence that generates photodisproportionated, metastable products, which are inferred to be Rh(III)L6 and Rh3(II,II,I)La These products have half-lives in the kilosecond range. The proposed photochemical processes thus achieve photoinduced charge separation, with remarkably long lifetimes for the charge-separated species. The metastable species may be viewed as a photogenerated electron-hole pair in solution that, due to its long lifetimes, may have significant potential in converting light into usable chemical energy.

When nitric oxide (NO) binds to heme proteins, it exerts a repulsive trans effect on the proximal ligand, resulting in weakening or rupture of the proximal ligand-iron bond. The general question of whether NO binding generates a five-coordinate complex with proximal ligand release is important for the function of enzymes such as guanylate cyclase. This question can be addressed by studying NO binding to the myoglobin cavity mutant H93G, where the proximal histidine has been replaced by glycine. When this protein is expressed in the presence of imidazole (Im), an imidazole molecule occupies the proximal cavity and serves as a ligand to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554]. This proximal imidazole can be exchanged for a variety of exogenous ligands [DePillis, G.D., Decatur, S. M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. While CO binds to H93G(Im) to form a stable six-coordinate complex similar to that of the wild type and NO binds to wild-type myoglobin to form a six-coordinate complex, we find that the binding of NO to H93G(Im) under similar conditions results in the cleavage of the exogenous imidazole-iron bond at neutral pH, leaving a five-coordinate heme-NO complex, H93G-NO, inside the protein. When a large excess of imidazole is added to this five-coordinate NO complex, a six-coordinate complex can be formed; thus, the binding constant of a sixth ligand to the five-coordinate H93G-NO complex can be measured. This is found to be several orders of magnitude smaller than the binding constant of Im to the carbonmonoxy, deoxy, or the metcyano forms of protein. By replacement of Im with methyl-substituted imidazoles which have hindered or strained binding conformations, this binding constant can be reduced further and some of the factors responsible for favoring the five-coordinate form can be elucidated. Thus, the cavity mutant H93G provides a novel model system for studying the factors that control the coordination state of NO complexes of heme proteins and serves as a bridge between synthetic heme model complexes in simple solvents and site-directed mutants in the structured environment found in proteins.

Ru2(O2C(CH2)6CH3)4 (1a) is soluble in both coordinating (THF, CH3OH, CH3CN) and noncoordinating solvents (benzene, toluene, cyclohexane, CH2Cl2), allowing its solution properties to be investigated by 1H and 13C NMR spectroscopy, UV/visible spectroscopy, resonance Raman spectroscopy, and cyclic voltammetry. In noncoordinating solvents, 1a exists as an oligomer, presumably by way of axial intermolecular -(- -[Ru2]- -O- -)n- interactions. 1H NMR studies of 1a and [Ru2(O2C(CH2)6CH3)4]+[X]-([1a]+[X]-), where X = Cl, BF4, or O2C(CH2)6CH3, indicate that both dipolar and contact mechanisms contribute to the paramagnetic shifts of the protons. Resonances for axial and equatorial ligands are shifted upfield and downfield, respectively, by a dipolar mechanism. Aromatic ligands in the axial sites, e.g. pyridine and pyrazine, experience an enhanced upfield shift by direct π-delocalization. Comparison of the 1H NMR signals for M2(O2CR)4 compounds where M = Ru and O2CR = benzoate, toluate, butyrate, crotonate, and dime...

Time-resolved infrared (TRIR) techniques have been employed to study the reactions of carbon monoxide with the cytochrome alpha 3-Cu(B) site of cytochrome c oxidase (CcO). The ligation dynamics immediately following photodissociation have been investigated using picosecond TRIR spectroscopy and linear dichroism. The rate of photoinitiated transfer of CO from cytochrome alpha 3 to CuB was measured directly by monitoring the development of the transient CuBCO absorption. In less than 1 ps, a stationary CuBCO spectrum develops, which together with the CO infrared linear dichroism is constant until the CO dissociates from CuB on a microsecond time scale. These observations indicate that the CO is transferred between metals and reaches its equilibrium conformation in less than 1 ps. This unprecedented ligand transfer rate has profound implications with regard to the structure and dynamics of the cytochrome alpha 3-CuB site, the functional architecture of the protein, and coordination dynamics in general.

The kinetics of the flash-induced photodissociation and rebinding of carbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectroscopy at room temperature and by Fourier transform infrared (FTIR) spectroscopy at low temperature. The binding of photodissociated CO to CuB+ at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the CuB(+)-CO complex. These measurements yield a first-order rate constant of (4.7 +/- 0.6) x 10(5) s-1 (t1/2 = 1.5 +/- 0.2 microseconds) for the dissociation of CO from the CuB(+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a(3)2+ exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the CuB(+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a(3)2+ (k2 = 1030 s-1). The CO transfer from CuB to Fe alpha 3 was followed by CO-FTIR between 158 and 179 K and by UV-vis at room temperature. The activation parameters over the temperature range 140-300 K are delta H++ = 10.0 kcal mol-1 and delta S++ = -12.0 cal mol-1 K-1. The value of delta H++ is temperature independent over this range; i.e., delta Cp++ = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a(3)2+ were observed. An increase in the alpha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initial femtosecond time-scale events accompanying photodissociation. Subsequently, a decrease is observed in the alpha-band absorbance (t1/2 approximately 1 microsecond) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by CuB+. We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the CuB(+)-CO complex. We suggest that the picosecond binding of photodissociated CO to CuB triggers the release of a ligand L from CuB. We infer that L then binds to cytochrome a3 on the distal side and that this process is directly responsible for the observed alpha-band absorbance changes. We have previously suggested that the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed.(ABSTRACT TRUNCATED AT 400 WORDS)

Results are presented which, taken with evidence developed by others, suggest a general mechanism for the entry and binding of exogenous ligands (including O2) at the "binuclear site" (CuBFea3) of the heme-copper oxidases. The mechanism includes a "ligand shuttle" wherein the obligatory way station for incoming ligands is CuB and the binding of exogenous ligands at this site triggers the exchange and displacement of endogenous ligands at Fea3. It is suggested that these ligand shuttle reactions might be functionally important in providing a coupling mechanism for electron transfer and proton translocation. Scenarios as to how this might happen are delineated.

Time-resolved resonance Raman (TR{sup 3}) spectra have been obtained for Re{sub 2}X{sub 8}{sup 2{minus}} (X = Cl, Br) in the A{sub 2u} ({delta}{delta}*) electronically excited state, at ambient temperature in solution. The Tr{sup 3} spectra exhibit Raman peaks that are assigned to the three symmetric vibrations of the excited state: the Re-Re stretch, Re-X stretch, and the Re-Re-X deformation. In addition, a depolarized peak attributed to an asymmetric X-Re-X bend is observed. Comparison of the TR{sub 3} results to single-crystal vibronic spectra reported by others clearly shows the effects of crystal constraints and observation time scale upon the strucutre of the {delta}{delta}* excited state. The TR{sup 3} data, obtained in solution on the nanosecond time scale, indicate that the excited state relaxes to a staggared molecular structure (D{sub 4} or D{sub 4d} symmetry). The vibronic data, obtained on single crystals under cryogenic conditions, are consistent with an eclipsed (D{sub 4h}) structure, similar to that of the ground state. A comparative TR{sup 3} study of quadruply bonded complexes, including both octahalodirhenate ions and Mo{sub 2}(PMe{sub 3}){sub 4}Cl{sub 4} (which is precluded by steric factors from significant torsional distorition about the metal-metal bond), was essential in elucidating the excited-state structures.

We report an investigation of complexes of the type M(2)(dmp)(4) (M = Mo, Cr; dmp = 2,6-dimethoxyphenyl) using resonance Raman (RR) spectroscopy, Cr isotopic substitution, and density functional theory (DFT) calculations. Assignment of the Mo-Mo stretching vibration in the Mo(2) species is straightforward, as evidenced by a single resonance-enhanced band at 424 cm(-1), consistent with an essentially unmixed metal-metal stretch, and overtones of this vibration. On the other hand, the Cr(2) congener has no obvious metal-metal stretching mode near 650-700 cm(-1), where empirical predictions based on the Cr-Cr distance as well as DFT calculations suggest that this vibration should appear if unmixed. Instead, three bands are observed at 345, 363, and 387 cm(-1) that (a) have relative RR intensities that are sensitive to the Raman excitation frequency, (b) exhibit overtones and combinations in the RR spectra, and (c) shift in frequency upon isotopic substitution ((50)Cr and (54)Cr). DFT calculations are used to model the vibrational data for the Mo(2) and Cr(2) systems. Both the DFT results and empirical predictions are in good agreement with experimental observations in the Mo(2) complex, but both, while mutually consistent, differ radically from experiment in the Cr(2) complex. Our experimental and theoretical results, especially the Cr isotope shifts, clearly demonstrate that the potential energy of the Cr-Cr stretching coordinate is distributed among several normal modes having both Cr-Cr and Cr-ligand character. The general significance of these results in interpreting spectroscopic observations in terms of the nature of metal-metal multiple bonding is discussed.

Far-infrared spectra are reported for mulls of the compounds Mo[sub 2]X[sub 4]L[sub 4] (X = Cl, Br, I; L = PMe[sub 3], PEt[sub 3], P(n-Pr)[sub 3], P(n-Bu)[sub 3], AsMe[sub 3]) and W[sub 2]Cl[sub 4](PMe[sub 3])[sub 4] and for a single crystal of Mo[sub 2]Cl[sub 4](PMe[sub 3])[sub 4]. Assignments have been made, using predictions of absorption intensities from a vector addition model and of frequencies by correlations to related molecules, of the [nu][sub 6] [b[sub 2], [nu](MX)], [nu][sub 8] [b[sub 2], [delta](MMX)], [nu][sub 10] [e, [nu](MX)], and [nu][sub 11] [e, [nu](MP)] modes. The spectra of Mo[sub 2]Cl[sub 4](PR[sub 3])[sub 4] complexes are complicated by the presence of internal ligand deformation modes in the same frequency region as [nu][sub 10]. 26 refs., 5 figs., 7 tabs.

The vibrational and electronic spectra of axially ligated sulfate-bridged binuclear platinum complexes ((Pt{sub 2}(SO{sub 4}){sub 4}L{sub 2}){sup n{minus}}: L = OH{sub 2}, DMSO, n = 2; L = Cl, Br, n = 4) have been studied. Raman spectra acquired with near-UV excitation show bands assigned to Pt-O(sulfate) stretching (300-350-cm{sup {minus}1} region) for all complexes, while bands assigned to Pt-Pt and pt-L stretching are seen for complexes (L = Cl, Br) that display preresonance Raman scattering within intense near-UV absorption bands assigned to the {sigma}(L) {r arrow} {sigma}{sup {star}}(Pt{sub 2}) electronic transition. Weaker electronic absorptions are assigned as metal-metal {sup 1}({sigma} {r arrow} {sigma} {sup {star}}) (near 220 nm for L =OH{sub 2}, Cl) and d{pi} {r arrow} d{sigma}{sup {star}}(Pt{sub 2}) (350-500 nm). Single-crystal polarized absorption spectra (L = OH{sub 2}, DMSO) show that the weak absorption system in the 350-500-nm region consists of three z-polarized (metal-metal axis) bands and an x,y-polarized band. The L = OH{sub 2}, Cl complexes show weak emission bands in the 695-750-nm region when excited at 77 K with near-UV light. These emission bands are similar to those observed for (Pt{sub 2}(HPO{sub 4}){sub 4}L{sub 2}){sup n{minus}} (L = OH{sub 2}, n = 2; L = Cl,more » n = 4) complexes. The emission lifetimes for the sulfate-bridged complexes (450 {plus minus} 50 ns, L = OH{sub 2}; 290 {plus minus} 20 ns, L = Cl) are much shorter, and the emission intensities are much weaker, than the corresponding properties of the phosphate-bridged complexes.« less

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (approximately 1 microseconds) as the post-photodissociation CU+B-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CuB to Fea3 when CO binds to CuB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CuB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fea3 pair. We speculate as to the identity of the ligand that is transferred between CuB and Fea3 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.

Time-resolved resonance Raman (TR{sup 3}) spectra have been obtained for Re{sub 2}X{sub 8}{sup 2{minus}} (X = Cl, Br) in the A{sub 2u} ({delta}{delta}*) electronically excited state, at ambient temperature in solution. The Tr{sup 3} spectra exhibit Raman peaks that are assigned to the three symmetric vibrations of the excited state: the Re-Re stretch, Re-X stretch, and the Re-Re-X deformation. In addition, a depolarized peak attributed to an asymmetric X-Re-X bend is observed. Comparison of the TR{sub 3} results to single-crystal vibronic spectra reported by others clearly shows the effects of crystal constraints and observation time scale upon the strucutre of the {delta}{delta}* excited state. The TR{sup 3} data, obtained in solution on the nanosecond time scale, indicate that the excited state relaxes to a staggared molecular structure (D{sub 4} or D{sub 4d} symmetry). The vibronic data, obtained on single crystals under cryogenic conditions, are consistent with an eclipsed (D{sub 4h}) structure, similar to that of the ground state. A comparative TR{sup 3} study of quadruply bonded complexes, including both octahalodirhenate ions and Mo{sub 2}(PMe{sub 3}){sub 4}Cl{sub 4} (which is precluded by steric factors from significant torsional distorition about the metal-metal bond), was essential in elucidating the excited-state structures.

We report the Stark spectra of the Soret, Q, and charge-transfer bands of deoxymyoglobin. The data show that band III has charge-transfer character but that the magnitude of the charge displacement is significantly smaller than expected or than what is observed for band I or even the Soret or Q bands. The data also show that symmetry breaking of iron−protoporphyrin IX in myoglobin produces a larger difference dipole moment in the Soret transition when compared to the dimethyl ester of protoporphyrin IX. The results lend further credence to the hypothesis that the electrostatic environment of the heme pocket in myoglobin is important in modifying the properties of the heme transitions, and they provide a basis for quantitative analysis of the static and time-dependent band shifts observed in response to environmental electrostatic perturbations and ligand binding dynamics.

Application of pressure (up to 150 kbar, where 1 kbar = 986.9 atm = 0.1 GPa) to solutions of Re{sub 2}X{sub 8}{sup 2{minus}} (X = F, Cl, Br) and Mo{sub 2}Cl{sub 4}(PMe{sub 3}){sub 4} results in smooth, continuous changes in experimental observables related to metal-metal bond length and, for the Re complexes, torsional angle (X-Re-Re-X dihedral angle). Resonance Raman studies show that increasing pressure engenders an increase in the frequency of the metal-metal stretching mode for all of the species studied, and this change is empirically correlated with a decrease in metal-metal bond length. Large red shifts in the energy of the {sup 1}({delta} {yields} {delta}*) transitions with increased pressure for Re{sub 2}Br{sub 8}{sup 2{minus}} and Re{sub 2}Cl{sub 8}{sup 2{minus}}, in contrast to the relative insensitivity of Re{sub 2}F{sub 8}{sup 2{minus}} absorption and Mo{sub 2}Cl{sub 4}(PMe{sub 3}){sub 4} fluorescence transitions, imply a tendency for the former complexes to distort torsionally from an eclipsed configuration toward a staggered configuration due to pressure-induced steric repulsion of the chloride and bromide ligands. Integrated absorption intensities further show that, at higher pressures, even Re{sub 2}F{sub 8}{sup 2{minus}} undergoes some torsional distortion, whereas the steric repulsion of adjacent phosphines located at a 90{degree} dihedral anglemore » on opposite Mo atoms prevents Mo{sub 2}Cl{sub 4}(PMe{sub 3}){sub 4} from distorting along the torsional coordinate. 28 refs., 7 figs., 1 tab.« less

The size and metabolic rate of cells affect processes from the molecular to the organismal level. We present a quantitative, theoretical framework for studying relationships among cell volume, cellular metabolic rate, body size, and whole-organism metabolic rate that helps reveal the feedback between these levels of organization. We use this framework to show that average cell volume and average cellular metabolic rate cannot both remain constant with changes in body size because of the well known body-size dependence of whole-organism metabolic rate. Based on empirical data compiled for 18 cell types in mammals, we find that many cell types, including erythrocytes, hepatocytes, fibroblasts, and epithelial cells, follow a strategy in which cellular metabolic rate is body size dependent and cell volume is body size invariant. We suggest that this scaling holds for all quickly dividing cells, and conversely, that slowly dividing cells are expected to follow a strategy in which cell volume is body size dependent and cellular metabolic rate is roughly invariant with body size. Data for slowly dividing neurons and adipocytes show that cell volume does indeed scale with body size. From these results, we argue that the particular strategy followed depends on the structural and functional properties of the cell type. We also discuss consequences of these two strategies for cell number and capillary densities. Our results and conceptual framework emphasize fundamental constraints that link the structure and function of cells to that of whole organisms.

In the time-resolved resonance Raman (TR3) spectrum of [Ru(phen)3]2+* (phen is 1,10-phenanthroline) in CH3CN at 298 K (shown here), ground-state bands dominate even at high laser irradiances. Howev...

Microperoxidase-11 has been immobilized on siliceous materials MCM-41 and SBA-15 and on amino-functionalized SBA-15. Resonance Raman spectroscopy has provided solid evidence that the exogenous species occupy the pores of the mesoporous silica materials. Photoreduction of the microperoxidase-11 Fe(III) center has been observed to occur in the immobilized samples and results in a long-lived stable reduced heme. Reoxidation of the heme occurs upon addition of oxygen, and the redox cycle can be repeated numerous times. The source of the electron resulting in reduction of the heme is proposed to originate from the silica matrix, and functionalization of silica surface is suggested to facilitate electron transfer to the heme.

We report CW-EPR, ESEEM, and structural NMR results, as well as DFT calculations, on model compounds relevant to the unusual cross-linked Tyr-His (YH) moiety at the active site of the heme-copper oxidases. CW-EPR spectra of an (15)N isotopically labeled 4-methyl-2-(4-methyl-imidazole-1-yl)-phenol radical are nearly identical to those of the natural abundance (14)N compound. We obtain good simulations of these EPR spectra without including hyperfine couplings to the nitrogen nuclei. This implies that the electron distribution of the radical is largely localized on the phenol ring with only a small amount of spin delocalized onto the nitrogens of the imidazole. Using three-pulse ESEEM spectroscopy, we have successfully detected the two imidazole ring nitrogens, one near the "exact cancellation" ESEEM condition and the other more weakly coupled. We assign these to the imino and amino nitrogens, respectively, based on DFT calculations performed on this radical species. The experimental results and the supporting density functional calculations clearly show that the imidazole substituent has only a minor effect on the electronic structure of the substituted phenol radical.

Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water and has been the subject of many studies. The focus of previous studies has been the characterization of intermediates that occur in the reduction of dioxygen to water. Cytochrome a3, one of the hemes in cytochrome c oxidase, contains a farnesyl tail which is though to ‘shuttle’ in and out of the binuclear site during the course of the catalytic reaction. While in the binuclear site, the tail is though to be coordinated to the heme iron center in a π-allyl fashion. With this though in mind, the synthesis and characterization of two model complexes for this π-allyl intermediate are reported here.

FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D(2)O (pH 6-9.3) between the MV-CO adduct (heme a(3) and Cu(B) reduced; heme a and Cu(A) oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a(3) oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a(3) decreases from 1965.3 cm(-1) at pH*

The fact that metabolic rate scales as the three-quarter power of body mass ( M ) in unicellular, as well as multicellular, organisms suggests that the same principles of biological design operate at multiple levels of organization. We use the framework of a general model of fractal-like distribution networks together with data on energy transformation in mammals to analyze and predict allometric scaling of aerobic metabolism over a remarkable 27 orders of magnitude in mass encompassing four levels of organization: individual organisms, single cells, intact mitochondria, and enzyme molecules. We show that, whereas rates of cellular metabolism in vivo scale as M −1/4 , rates for cells in culture converge to a single predicted value for all mammals regardless of size. Furthermore, a single three-quarter power allometric scaling law characterizes the basal metabolic rates of isolated mammalian cells, mitochondria, and molecules of the respiratory complex; this overlaps with and is indistinguishable from the scaling relationship for unicellular organisms. This observation suggests that aerobic energy transformation at all levels of biological organization is limited by the transport of materials through hierarchical fractal-like networks with the properties specified by the model. We show how the mass of the smallest mammal can be calculated (≈1 g), and the observed numbers and densities of mitochondria and respiratory complexes in mammalian cells can be understood. Extending theoretical and empirical analyses of scaling to suborganismal levels potentially has important implications for cellular structure and function as well as for the metabolic basis of aging.

The electronic structure of the binuclear copper complex [Cu(2)(L)](3+) [L = N(CH(2)CH(2)N(H)CH(2)CH(2)N(H)CH(2)CH(2))(3)N] has been investigated by resonance Raman and electroabsorption spectroscopy. Crystallographic Cu(2) distances of 2.364(1) and 2.415(1) A determined for the nitrate and acetate salts, respectively, are consistent with a substantial metal-metal interaction. The Cu-Cu bonding interaction in the binuclear complex is modulated both in the solid state and in solution by the ligand environment through coupling to ligand torsional modes that are, in turn, stabilized by hydrogen bonding. Electroabsorption data on the three major visible and near-infrared electronic transitions of Cu(2)L, lambda(max) (epsilon(max)) = 1000 nm ( approximately 1200 M(-1) cm(-1)), 748 nm (5600 M(-1) cm(-1)), and 622 nm (3350 M(-1) cm(-1)), reveal a difference dipole moment between the ground and excited states (Deltamu(A)) because of symmetry breaking. The difference polarizability for all three of the transitions is negative, indicating that the ground state is more polarizable than the excited state. A general model to explain this behavior in terms of the proximity of accessible transitions involving copper d electrons is proposed to explain the larger polarizability of the ground state. Raman excitation profiles (REPs) provide evidence for multiple conformational states of [Cu(2)(L)](3+). Separate REPs were obtained for each of the components of the two major Raman bands for nu(1) (a Cu-Cu stretching mode) and nu(2) (a Cu-Cu-N(eq) bending mode). The Raman data along with quantum chemical ZINDO/S CI calculations provide evidence for isomeric forms of Cu(2)L with strong coupling between the conformation of L and the Cu-Cu bond length.

Solution (VT NMR, Evans method magnetic susceptibility, resonance Raman) and solid-state (SQUID magnetic susceptibility, X-ray crystallography) spectroscopic studies of intertriad heterodimeric [(OEP)MoRu(OEP)] (1), [(OEP)WOs(OEP)] (2), and [(OEP)MoRu(TPP)]PF(6) (3(+)) metalloporphyrins are reported (OEP = 2,3,7,8,12,13,17,18-octaethylporphyrinato; TPP = 5,10,15,20-tetraphenylporphyrinato). Solution and solid-state magnetic susceptibility data indicate that 1 and 2 contain two unpaired electrons in the ground electronic configuration. The presence of a delta bond in 3(+) has been confirmed by structural characterization. The experimental evidence is consistent with a molecular orbital ordering, sigmapideltapidelta, which is different from that seen for homologous metalloporphyrin dimers with homometallic or intratriad heterometallic multiple metal-metal bonds. Resonance Raman data suggest that the heterometallic bonds are slightly stronger than isoelectronic homometallic species.

Solution ((1)H NMR, Evans method magnetic susceptibility, resonance Raman) and X-ray crystallographic spectroscopic studies of intertriad heterodimeric [(OEP)MoOs(OEP)] (3), [(OEP)WRu(OEP)] (4), [(OEP)MoOs(TPP)]PF(6) (5(+)), and [(OEP)WRu(TPP)]PF(6) (6(+)) metalloporphyrins are reported (OEP = 2,3,7,8,12,13,17,18-octaethylporphyrinato; TPP = 5,10,15,20-tetraphenylporphyrinato). Evans method magnetic susceptibility data indicate that 3 and 4 contain two unpaired electrons in the ground electronic configuration. Resonance Raman spectra of 3, 4, 5(+), and 6(+) suggest that WRu bonds are 5-10% stronger than corresponding MoOs species. Structural characterization of 5(+) and 6(+) demonstrates metal-metal bond lengths of 2.30 (WRu) and 2.24 (MoOs) Å, respectively. The possibility of a special stability associated with polar heterometallic multiple bonds is discussed.

Cytochrome c oxidase (EC 1.9.3.1), the oxygen-reducing member of the respiratory chains of mitochondria and several aerobic bacteria, catalyzes flux of reducing equivalents to the highly oxidizing O2-H2O couple and conserves energy as a proton-motive force. These enzymes are members of a large family of related heme-copper oxidases; others are the hydroquinone oxidases, of which cytochrome bo 3 of Escherichia coli is the best characterized. Apart from the intriguing catalysis of O2 reduction to water, this class of enzymes conserves energy by vectorial functioning as proton and electrical charge trans locators. This leads to primary conservation of respiratory redox energy as an electrochemical proton gradient across the mitochondrial or bacterial membrane, which is secondarily utilized as the driving force for the oxidative synthesis of ATP (Mitchell, 1966). Before the elucidation of the crystal structures of cytochrome c oxidase from Paracoccus denitrificans (Iwata et al., 1995) and bovine heart mitochondria (Tsukihara et al., 1995, 1996), the heme-copper oxidases have been the subject of many review articles, which have highlighted their functional (Malmstrom, 1990a,b; Cooper, 1990; Chan and Li, 1990; Wilson and Bickar, 1991; Rich, 1991; Babcock and Wikstrom, 1992), structural (Capaldi, 1990; Saraste, 1990; Saraste et al., 1991; Gennis, 1992; Haltia and Wikstrom, 1992; Hosler et al., 1993), and evolutionary aspects (Kadenbach et al., 1987, 1991; Castresana et al., 1995; Saraste et al., 1996). The present account is not intended as a review.

Photodissociation of fully reduced, carbonmonoxy cytochrome bo3 causes ultrafast transfer of carbon monoxide (C triple bond O) from heme iron to CuB in the binuclear site. At low temperatures, the C triple bond O remains bound to CuB for extended times. Here, we show that the binding of C triple bond O to CuB perturbs the IR stretch of an un-ionized carboxylic acid residue, which is identified as Glu286 by mutation to Asp or to Cys. Before photodissociation, the carbonyl (C=O)-stretching frequency of this carboxylic acid residue is 1726 cm-1 for Glu286 and 1759 cm-1 for Glu286Asp. These frequencies are definitive evidence for un-ionized R-COOH and suggest that the carboxylic acids are hydrogen-bonded, though more extensively in Glu286. In Glu286Cys, this IR feature is lost altogether. We ascribe the frequency shifts in the C=O IR absorptions to the effects of binding photodissociated C triple bond O to CuB, which are relay ed to the 286 locus. Conversely, the 2065 cm-1 C triple bond O stretch of CuB-CO is markedly affected by both mutations. These effects are ascribed to changes in the Lewis acidity of CuB, or to displacement of a CuB histidine ligand by C triple bond O. C triple bond O binding to CuB also induces a downshift of an IR band which can be attributed to an aromatic C-H stretch, possibly of histidine imidazole, at about 3140 cm-1. The results suggest an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286. A chain of bound water molecules may provide such a connection, which is of interest in the context of the proton pump mechanism of the heme-copper oxidases.

We address the molecular mechanism by which the haem-copper oxidases translocate protons. Reduction of O2 to water takes place at a haem iron-copper (CuB) centre, and protons enter from one side of the membrane through a ‘channel’ structure in the enzyme. Statistical-mechanical calculations predict bound water molecules within this channel, and mutagenesis experiments show that breaking this water structure impedes proton translocation. Hydrogen-bonded water molecules connect the channel further via a conserved glutamic acid residue to a histidine ligand of CuB. The glutamic acid side chain may have to move during proton transfer because proton translocation is abolished if it is forced to interact with a nearby lysine or arginine. Perturbing the CuB ligand structure shifts an infrared mode that may be ascribed to the O H stretch of bound water. This is sensitive to mutations of the glutamic acid, supporting its connectivity to the histidine. These results suggest key roles of bound water, the glutamic acid and the histidine copper ligand in the mechanism of proton translocation.

We report the fast relaxation dynamics of “native” apomyoglobin (pH 5.3) following a 10-ns, laser-induced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales (ν = 1633 cm −1 , τ = 48 ns; ν = 1650 cm −1 , τ = 132 μs). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm −1 ) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm −1 ). Folding rate coefficients inferred from the observed relaxations at 60°C are k f(solvated) = (7 to 20) × 10 6 s −1 and k f(native) = 3.6 × 10 3 s −1 , respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60°C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.

Resonance Raman spectra of the cubic metal-halide complexes having the general formula [M(6)X(8)Y(6)](2)(-) (M = Mo or W; X, Y = Cl, Br, or I) are reported. The three totally symmetric fundamental vibrations of these complexes are identified. The extensive mixing of the symmetry coordinates that compose the symmetric normal modes expected in these systems is not observed. Instead the "group-frequency" approximation is valid. Furthermore, the force constants of both the apical and face-bridging metal-halide bonds are insensitive to the identity of either the metal or the halide. Raman spectra of related complexes with methoxy and benzenethiol groups as ligands are reported along with the structural data for [Mo(6)Cl(8)(SPh)(6)][NBu(4)](2). Crystal data for [Mo(6)Cl(8)(SPh)(6)][NBu(4)](2) at -156 degrees C: monoclinic space group P2(1)/c; a = 12.588(3), b = 17.471(5), c = 20.646(2) Å; beta = 118.53(1) degrees, V = 3223.4 Å(3); d(calcd) = 1.664 g cm(-)(3); Z = 2.

A high-pressure stopped-flow apparatus developed in our laboratories provides the capability to use dissolved gaseous reactants at elevated concentrations in solution (in equilibrium with gas pressures up to ca. 30 atm) for measurement of reaction kinetics. We have used this apparatus to follow the reaction of dioxygen with bovine cytochrome c oxidase following photolysis of the fully reduced CO ligated enzyme up to a dioxygen concentration of 16 mM. The observed rate dependence on [O2] follows saturation kinetics and was fit to a limiting rate of 1.0 × 106s−1. This value is approximately the same as that for the thermal loss of CO to solution from the transient CuBbound state formed upon photolysis of the heme-CO complex. Implications for the mechanism of O2binding and reduction by the heme-copper oxidases are discussed.

The present work employs far-red resonance Raman spectroscopy (RR) to investigate whether a Cu-Cu bonding interaction exists in Cu{sub A}. The electronic transition of Cu{sub A} near 830 nm provides the opportunity for specific RR observation of the vibrations of this chromophore. We have employed RR with Cu isotopic substitution on genetically modified, solubilized forms of CcO subunit II from Bacillus subtilis and Paracoccus denitrificans. We have also probed the Cu{sub A} site of native beef heart CcO for comparison. The similarity of the CcO and Cu{sub A} fragment (subunit II) spectra suggests that Cu{sub A} in the isolated subunit II has essentially the same structure as that of Cu{sub A} in intact CcO. Thus, the suggested Cu-Cu bonding in Cu{sub A}, if correct, applies both to the isolated subunit II and native CcO. The functional reasons why nature may choose such a structure, or in fact why a two-copper center of any sort is constructed to perform a function which is commonly believed to be simple one-electron transfer, are unclear. 34 refs., 1 fig., 1 tab.