Detection and characterization of hyperfine-shifted resonances in the proton nuclear magnetic resonance spectrum of Anabaena 7120 ferredoxin at high magnetic fields

This paper presents previously unobserved signals in the 1H NMR spectra of oxidized and reduced [2Fe-2S]-ferredoxin from Anabaena 7120 detected at 400, 500, and 600 MHz. The signals shifted to low field exhibited longitudinal relaxation (T1) values in the range of 100-400 microseconds and line widths in the range of 1-10 kHz (at 400 MHz), and the chemical shifts of all signals showed strong temperature dependence. Although the line widths were smaller at lower magnetic fields, the resolution was better at higher magnetic fields. In the oxidized state, a broad signal was detected at 37 ppm, which corresponds to at least 6 protons, and whose chemical shift exhibits positive temperature dependence. This signal also was found in oxidized ferredoxin reconstituted in 2H2O, which excludes the signal as arising from solvent-exchangeable amide protons. In the reduced state, four signals detected between 90 and 140 ppm exhibited negative temperature dependence. These consisted of two pairs of signals, each pair having one component with half the linewidth of the other. On the basis of their chemical shifts, linewidths, longitudinal relaxation properties, and temperature dependence we assigned these resonances to four of the beta hydrogens of the ligated cysteines. Two solvent-exchangeable hyperfine-shifted signals were found in the reduced state; these are located upfield of the diamagnetic region. The low-field hyperfine resonances of half-reduced ferredoxin in the presence of sodium dithionite showed a self electron transfer exchange rate that was slow on the NMR scale as observed earlier (Chan, T., and Markley, J. L. (1983) Biochemistry 22, 5982-5987), but the exchange rate was accelerated in the presence of methyl viologen.     

Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances

Proton NMR spectra of the oxidized and reduced forms of high-potential iron-sulfur proteins (HiPIPs) were recorded at 200 MHz. The proteins studied were the HiPIPs I and II from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata. Hyperfine-shifted peaks in spectra of the oxidized proteins were assigned to some of the protons of the cysteinyl ligands and aromatic residues at the active site on the basis of their chemical shifts, longitudinal relaxation times, and temperature-dependent behavior. The cysteinyl C beta-H protons were found to resonate downfield (about 100 ppm) and the C alpha-H protons upfield (about-25 ppm). This hyperfine shift pattern is consistent with the observed isotropic shift being contact in origin; it probably results from a pi-spin-transfer mechanism. The large magnitudes of the chemical shifts of peaks assigned to aromatic residues suggest that these residues interact with the iron-sulfur cluster via pi-pi overlap. Some of the hyperfine-shifted peaks observed in water were found to disappear in 2H2O solution. Such resonances probably arise from exchange-labile hydrogens of amino acid residues directly hydrogen bonded to the iron-sulfur cluster. In the case of HiPIPs I and II from E. vacuolata, whose spectra are similar except for the number of such peaks, the relative number of hydrogen bonds inferred to be present in the oxidized and reduced proteins qualitatively explains the difference between their midpoint redox potentials. On the other hand, for E. halophila HiPIPs I and II, consideration of the inferred number of hydrogen bonds alone fails to predict the sign of the difference between their midpoint redox potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton magnetic resonance spectra of adrenodoxin: features of the aromatic region

This paper presents the first 1H-NMR spectra of the aromatic region of adrenodoxin, a mammalian mitochondrial 2Fe-2S non-heme iron ferredoxin. One-dimensional proton NMR spectra of both reduced and oxidized adrenodoxin were recorded as a function of pH. Resonances due to two of the three histidines of adrenodoxin gave sharp signals in the one-dimensional proton NMR spectra. The pKa values of the resolved histidine resonances in the oxidized protein were 6.64 +/- 0.03 and 6.12 +/- 0.06. These values were unchanged when adrenodoxin was reduced by the addition of sodium dithionite. In addition, the oxidized protein showed a broadened histidine C-2H resonance with a pKa value of approx. 7. This resonance was not apparent in the spectra of the reduced protein. The resonances due to the single tyrosine in adrenodoxin were identified using convolution difference spectroscopy. In addition, a two-dimensional Fourier-transform double quantum filtered (proton, proton) chemical shift correlated (DQF-COSY) spectrum of oxidized adrenodoxin was obtained. The cross peaks of the resonances due to the tyrosine, the four phenylalanines, and two of the three histidines of adrenodoxin were resolved in the DQF-COSY spectrum. Reduction of the protein caused several changes in the aromatic region of the NMR spectra. The resonances assigned to the C2 proton of the histidine with a pKa of 6.6 shifted upfield approx. 0.15 ppm. In addition, when the protein was reduced one of the resonances assigned to a phenylalanine residue with a chemical shift of 7.50 ppm appeared to move downfield to 7.82 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spinach plastocyanin: Comparison of reduced and oxidized forms by natural abundance carbon-13 nuclear magnetic resonance spectroscopy

Differences between the reduced Cu(I) and oxidized Cu(II) forms of spinach plastocyanin were investigated by natural abundance carbon-13 nuclear magnetic resonance spectroscopy at 67.9 MHz using proton noise decoupling. The spectra confirm that histidines 38 and 91 are copper ligands and demonstrate that coordination is by the N^o1 of both imidazole rings. Spectra of reduced plastocyanin yielded 128 separately resolved carbon resonances. Upon oxidation, 16 of these were observed to disappear; yet there was little change in the positions or intensities of other peaks. Those peaks which disappear are assigned to carbons near the metal. The protein evidently does not undergo a substantial change in conformation upon change of redox state.     

1H NMR studies of porcine uteroferrin. Magnetic interactions and active site structure

Pink (reduced) uteroferrin exhibits well resolved paramagnetic NMR spectra with resonances ranging from 90 ppm downfield to 70 ppm upfield. The intensities of these signals depend on the degree of reduction and correlate well with the intensity of the EPR signals with gave = 1.74. Analyses of chemical shifts and the temperature dependence of the paramagnetically shifted resonances indicate that the Fe(III)-Fe(II) cluster in the reduced protein exhibits weak antiferromagnetic exchange coupling (-J approximately equal to 10 cm-1), in agreement with the estimate derived from the temperature dependence of the EPR signal intensity. Purple (oxidized) uteroferrin, on the other hand, exhibits no discernible paramagnetically shifted resonances, reflecting either strong antiferromagnetic coupling or an unfavorable electron spin-lattice relaxation time. Evans susceptibility comparisons between pink and purple uteroferrin show that the Fe(III)-Fe(III) cluster in the oxidized protein is more strongly coupled (-J greater than 40 cm-1). This value concurs with low temperature magnetic susceptibility measurements on both the porcine and splenic purple acid phosphatases. The isotropically shifted protons of tyrosine coordinated to the cluster are assigned by comparison with synthetic complexes. Tyrosine, earlier implicated as a ligand by resonance Raman spectroscopy, appears to coordinate only to the ferric site in pink uteroferrin. This is consistent with the relatively invariant extinction coefficients of uteroferrin in its oxidized and reduced forms and the ease of reduction of the nonchromophoric iron compared to its chromophoric partner. Other possible ligands to the cluster include histidine, suggested by the presence of downfield-shifted solvent-exchangeable resonances with appropriate isotropic shifts.     

Proton nuclear magnetic resonance spectra of Pseudomonas aeruginosa ferricytochrome cd1

Proton nuclear magnetic resonance spectra of ferricytochrome cd1 from the denitrifying bacterium Pseudomonas aeruginosa have been obtained. The normal 0-10-ppm chemical shift range shows many overlapping and nonresolvable peaks, as would be expected for a dimeric protein of molecular weight approximately 120,000. In the downfield region between 10 and 50 ppm, and in the upfield region between 0 and -20 ppm, resolvable resonances corresponding to a small number of protons are observed. The temperature and pH behavior of these resonances have been examined. For some of the resolved resonances, the pH behavior of chemical shifts and intensities indicates that the oxidized form of the enzyme undergoes a structural transition with a pK of 5.8 +/- 0.3. On the basis of several lines of evidence, some assignments are proposed in which resolvable resonances are assigned as originating from either the heme c or the heme d1 prosthetic groups of the enzyme.     

1H-NMR studies of high-potential iron-sulfur protein from the purple phototrophic bacterium, Rhodospirillum tenue

The high-potential iron-sulfur protein (HiPIP) from Rhodospirillum tenue (strain 3761) shows only a weak (20-25%) sequence similarity to HiPIPs from Chromatium vinosum, Ectothiorhodospira halophila and Ectothiorhodospira vacuolata, including the strict conservation of only two of the twelve residues assumed to be in the 4Fe-4S cluster packing region [Tedro, S. M., Meyer, T. E. and Kamen, M. D. (1979) J. Biol. Chem. 254, 1495-1500]. In spite of these differences, the general range and distribution of hyperfine-shifted 1H-NMR peaks of oxidized and reduced R. tenue HiPIP resemble those of E. halophila HiPIP I [Krishnamoorthi, R., Markley, J. L., Cusanovich, M. A., Pryzycieki, C. T. and Meyer, T. E. (1986) Biochemistry 25, 60-67]. Temperature- and pH-dependence and longitudinal relaxation behavior were determined for hyperfine-shifted peaks of the oxidized protein. Tentative assignments of peaks to ligands and aromatic residues suggest the presence of common apoprotein-active-site interactions in these proteins. Differences occur in the pattern of paramagnetically shifted peaks attributed to hydrogens bonded to the 4Fe-4S cluster. Hyperfine-shifted peaks of R. tenue HiPIP are not perturbed by pH changes in the range 5-9. In contrast, those of the C. vinosum protein exhibit a pH-dependence of chemical shifts that has been attributed to the titration of His42 [Nettesheim, D. G., Meyer, T. E., Feinberg, B. A. and Otvos, J. D. (1983) J. Biol. Chem. 258, 8235-8239]. Since R. tenue HiPIP contains no histidine, the present observation confirms the above hypothesis.     

Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the 2Fe-2S* cluster

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change of adrenodoxin induced by reduction of iron-sulfur cluster. Proton nuclear magnetic resonance study.

Bovine adrenodoxin in the reduced form has been measured by one- and two-dimensional 1H NMR spectroscopy. By comparing the spectrum of reduced adrenodoxin with that of the oxidized protein, resonances have been assigned for the aromatic residues. The spin-lattice relaxation time for the resonances due to histidine residues was found to depend on the reduction state of adrenodoxin. The distance from the paramagnetic center is calculated by using the Solomone-Bloembergen equation. The resonances from Tyr-82 and Ala-81 show large chemical shift changes upon reduction of adrenodoxin. The conformational change of adrenodoxin manifested by chemical shift difference between reduced and oxidized forms is found in the sequence around Tyr-82 and Ala-81. Modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at Glu-74, Asp-79, and Asp-86 inhibited the interaction with both adrenodoxin reductase and cytochrome P-450scc (Lambeth, D. J., Geren, L. M., and Millett, F. (1984) J. Biol. Chem. 259, 10025-10029; Geren, L. M., O'Brien, P., Stonehuerner, J., and Millett, F. (1984) J. Biol. Chem. 259, 2155-2160). Thus, the sequence of these amino acids was assigned to the interaction site with the redox partners. The present 1H NMR investigation of adrenodoxin demonstrates that a conformational change upon reduction of the iron-sulfur cluster occurs in the sequence of negatively charged amino acids that is a putative site for interaction with redox partners. This could offer the structural basis of the electron transfer mechanism in which adrenodoxin functions as a mobile electron carrier.     

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.     

Theory of relaxation of mobile water protons induced by protein NH moieties, with application to rat heart muscle and calf lens homogenates.

Kimmich and co-workers (cf., Winter, F., and R. Kimmich. 1982. Biochim. Biophys. Acta. 719:292-298) discovered peaks in the magnetic field-dependent longitudinal relaxation rate (1/T1) of water protons of muscle tissue, cells, and dehydrated protein in the field range 0.5-5 MHz (proton Larmor frequency), and argued that the peaks resulted from cross relaxation associated with quadrupolar splittings of the 14N nuclei of protein NH groups. More recently, analogous peaks were found in homogenates of calf eye lens (Beaulieu, C.F., J.I. Clark, R.D. Brown III, M. Spiller, and S. H. Koenig, 1987. Abstr. Soc. Magn. Res. Med., 6th, New York. 598-599), which are essentially concentrated protein solutions, and were measured with sufficient precision to allow resolution of the relaxation spectra into several peaks and the intrinsic linewidths to be determined. Here, we analyze these relaxation spectra, as well as earlier data on rat heart (Koenig, S. H., R. D. Brown III, D. Adams, D. Emerson, and C. G. Harrison. 1984. Invest. Radiol. 19:76-81) in some detail, and suggest a specific pathway for the cross relaxation to which we apply the theory of relaxation quantitatively. The view that emerges is that, at fields such that the proton Zeeman energy of the NH protons matches an 14N quadrupolar splitting, relaxation of these protons is by cross relaxation to the 14N nuclei which in turn transfer excess energy to the protein. The correlation time for the NH proton interaction is the T2 of the 14N nuclei, approximately 10(-6) s, whereas T1 of the NH protons is approximately 1.25 ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton resonance assignments of horse ferricytochrome c

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.     

Advances towards resonance assignments for uniformly—13C, 15N enriched bacteriorhodopsin at 18.8 T in purple membranes

Solid state NMR spectra from uniformly (13)C, (15)N enriched bacteriorhodospin (bR) purified from H. salinarium were acquired at 18.8 T using magic angle spinning methods. Isolated resonances of 2D (13)C-(13)C spectra exhibited 0.50-0.55 ppm line-widths. Several amino acid types could be assigned, and at least 12 out of 15 Ile peaks could be resolved clearly and identified based on their characteristic chemical shifts and connectivities. This study confirms that high resolution solid state NMR spectra can be obtained for a 248 amino acid uniformly labeled membrane protein in its native membrane environment and indicates that site-specific assignments are likely to be feasible with heteronuclear multidimensional spectra.     

15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c

Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz. The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints. Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein. By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein. The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states. The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility. Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.     

Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.     

Distance measurements in spin-labeled lysozyme

The single His-15 of hen egg lysozyme reacts with 2,2,6,6-tetramethyl-4-(bromoacetamido)piperidinyl-1-oxy or 2,2,5,5-tetramethyl-3-(bromoacetamido)pyrrolidinyl-1-oxy to give a spin-labeled enzyme [Wien, R. W., Morrisett, J. D., & McConnell, H. M. (1972) Biochemistry 11, 3707-3716]. High-field 1H NMR spectra (300 and 500 MHz) of these species in 2H2O contain protein peaks selectively broadened by dipolar coupling to the unpaired electron spin. While usually difficult to discern in the spectrum itself, broadened resonances are revealed in difference spectra obtained by subtracting the original spectrum from one taken after reduction of the nitroxide radical with ascorbate. The heights of difference spectra peaks are related in a simple way to r-6, where r is the label to proton distance. These distances were used to solve for the location of the electron spin by using algorithms from distance geometry. The spin was found to lie in a hydrophobic groove between Phe-3 and Asp-87. These results demonstrate the feasibility of spin-labeling for accurate distance measurements in proteins through the use of distance geometry.     

Rubredoxin from the green sulfur bacterium Chlorobium tepidum functions as an electron acceptor for pyruvate ferredoxin oxidoreductase.

Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of pyruvate to acetyl-CoA and CO(2), exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the pyruvate synthase or CO(2) fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOR/pyruvate synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mM(-1) cm(-1) with an A(492)/A(280) ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be -87 mV, which is the most electronegative value reported for Rd from any source.     

19F NMR studies on 8-fluoroflavins and 8-fluoro flavoproteins

The 19F NMR spectra of the oxidized and reduced forms of 8-fluororiboflavin, 8-fluoro-FAD, and the 8-fluoroflavin-reconstituted flavoproteins flavodoxin, riboflavin binding protein, D-amino acid oxidase, p-hydroxybenzoate hydroxylase, Old Yellow Enzyme, anthranilate hydroxylase, general acyl-CoA dehydrogenase, glucose oxidase, and L-lactate oxidase were measured. For the proteins studied the oxidized resonances appeared over a 10.1-ppm range, while the reduced resonances were spread over 10.3 ppm. Reduction caused an upfield shift of about 27 ppm for the free 8-fluoroflavins and most of the 8-fluoro flavoproteins. The notable exception was 8-fluoro-FMN flavodoxin, which was shifted 37.6 ppm, indicating an unusually high electron density in the benzene ring. Ligand binding to the oxidized 8-fluoro flavoproteins caused either upfield or downfield shifts of 1.5-5 ppm, depending on the protein/ligand combination. The 8-fluoro-FAD anthranilate hydroxylase resonance was shifted downfield and split into two peaks in the presence of anthranilate. The 8-fluoro-FMN Old Yellow Enzyme resonance was shifted upfield upon complexation with charge-transfer-forming, para-substituted phenolates. The upfield shift increased from less than 1 to 5 ppm as the electron-donating capacity of the phenolate increased. Complexation of native Old Yellow Enzyme with 2,4-difluorophenol caused the fluorine resonances of the ligand to shift and split into two pairs of signals. Each pair of signals was associated with a different isozyme of Old Yellow Enzyme.     

15N resonance assignments of oxidized and reducedChromatium vinosum high-potential iron protein

The¹⁵N resonances in reduced and oxidizedChromatium vinosum high-potential iron protein have been assigned by use of¹H-¹H COSY spectra and¹H-¹⁵N HMQC, HMQC-COSY, and HMQC-NOESY spectra. Unambiguous assignment of 70 of 85 backbone¹⁵N resonances in the reduced protein and 62 of 85 resonances in the oxidized protein are made, as are 12 of 21 side-chain¹⁵N resonances.