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• W2080290351 abstract "The heme ligation in the isolated cdomain of Paracoccus pantotrophus cytochromecd 1 nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochromecd 1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd 1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolatedc domain is 320.9 ± 1.2 K in the oxidized and 357.7 ± 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the cheme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of thed 1 heme in guiding His17 to thec heme in the oxidized holoenzyme is discussed. The heme ligation in the isolated cdomain of Paracoccus pantotrophus cytochromecd 1 nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochromecd 1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd 1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolatedc domain is 320.9 ± 1.2 K in the oxidized and 357.7 ± 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the cheme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of thed 1 heme in guiding His17 to thec heme in the oxidized holoenzyme is discussed. polyacrylamide gel electrophoresis chemical shift (ppm) heat capacity change upon denaturation difference in chemical shift enthalpy change upon unfolding at the midpoint of unfolding driven equilibrium Fourier transform sodium 2,2-dimethyl-2-silapentane-5-sulfonate double-quantum filtered coherence spectroscopy exchange spectroscopy matrix-assisted laser desorption/ionization time-of-flight nuclear Overhauser enhancement nuclear Overhauser enhancement spectroscopy glass-electrode reading of the pH meter at room temperature, uncorrected for deuterium isotope effects absolute temperature spin-lattice relaxation time temperature at the midpoint of unfolding total correlation spectroscopy The x-ray structures of Paracoccus pantotrophus(formerly Thiosphaera pantotropha (1Rainey F.A. Kelly D.P. Stackebrandt E. Burghardt J. Hiraishi A. Katayama Y. Wood A.P. Int. J. Syst. Bacteriol. 1999; 49: 645-651Crossref PubMed Scopus (141) Google Scholar)) cytochromecd 1 nitrite reductase in the oxidized and reduced forms have revealed a remarkable heme-ligand switching event at both hemes of this enzyme (2Williams P.A. Fülöp V. Garman E.F. Saunders N.F.W. Ferguson S.J. Hajdu J. Nature. 1997; 389: 406-412Crossref PubMed Scopus (254) Google Scholar). Upon reduction of thecd 1 enzyme in the crystal, Tyr25, which ligates the d 1 heme in the active site of the oxidized protein is released to allow substrate binding. Concomitantly, the c domain refolds, resulting in a change in c heme coordination from His17-His69 to His69-Met106 (see Fig. 1, see also Ref. 2Williams P.A. Fülöp V. Garman E.F. Saunders N.F.W. Ferguson S.J. Hajdu J. Nature. 1997; 389: 406-412Crossref PubMed Scopus (254) Google Scholar). During the conversion of nitrite to nitric oxide, both the cheme, which is the site of electron entry and thed 1 heme, the site where catalysis takes place, of cd 1 nitrite reductase are sequentially oxidized and the enzyme returns to its His17-His69 ligated oxidized state in the crystal when all reducing equivalents are exhausted. Similar heme-ligand switching has been observed since in other systems. The transcriptional activator CooA from Rhodospirillum rubrum contains a b-type heme that acts as a CO sensor in vivo. Under physiological conditions, CO can easily replace one of the ligands to the ferrous heme, thereby causing conformational changes around the heme and triggering activation of the transcriptional regulator (3Aono S. Ohkubo K. Matsuo T. Nakajima H. J. Biol. Chem. 1998; 273: 25757-25764Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The heme in CooA is in the six-coordinate form in both oxidation states, with Cys75being a heme ligand in the oxidized form (E 0 = −320 mV), whereas His77 appears to be an axial ligand in the reduced protein (E 0 = −260 mV) and is essential for activation of the protein by CO (3Aono S. Ohkubo K. Matsuo T. Nakajima H. J. Biol. Chem. 1998; 273: 25757-25764Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 4Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (99) Google Scholar, 5Aono S. Nakajima H. Coordin. Chem. Rev. 1999; 192: 267-282Crossref Scopus (36) Google Scholar, 6Nakajima H. Aono S. Chem. Lett. 1999; 11: 1233-1234Crossref Scopus (14) Google Scholar). Another example of a redox-driven heme-ligand switching event has been reported for the yeast F82H/C102S iso-1-cytochrome c variant (7Schejter A. Taler G. Navon G. Liu X.J. Margoliash E. J. Am. Chem. Soc. 1996; 118: 477-478Crossref Scopus (24) Google Scholar, 8Feinberg B.A. Liu X. Ryan M.D. Schejter A. Zhang C. Margoliash E. Biochemistry. 1998; 37: 13091-13101Crossref PubMed Scopus (46) Google Scholar). In the reduced state of the protein, the heme iron is coordinated by His18 and Met80, similar to the wild-type protein, whereas the heme iron is ligated by His18 and His82 in the oxidized protein. The redox potential of the His-Met form is E 0 = 247 mV and that of the His-His form is E 0 = 47 mV. Electrochemical examination has revealed that the oxidized His18-His82 form is highly disordered, and it is proposed that this high level of disorder facilitates rapid rearrangement to His18-Met80 upon reduction (8Feinberg B.A. Liu X. Ryan M.D. Schejter A. Zhang C. Margoliash E. Biochemistry. 1998; 37: 13091-13101Crossref PubMed Scopus (46) Google Scholar). Heme-ligand exchange reactions between methionine and histidine residues were first observed during the in vitro folding and unfolding reactions of oxidized (9Yeh S.R. Takahashi S. Fan B. Roussseau D.L. Nat. Struct. Biol. 1997; 4: 51-56Crossref PubMed Scopus (134) Google Scholar, 10Takahashi S. Yeh S.-R. Das T.K. Chan C.-K. Gottfried D.S. Rousseau D.L. Nat. Struct. Biol. 1997; 4: 44-50Crossref PubMed Scopus (216) Google Scholar, 11Yeh S.R. Rousseau D.L. J. Biol. Chem. 1999; 274: 17853-17859Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and reduced (12Telford J.R. Tezcan F.A. Gray H.B. Winkler J.R. Biochemistry. 1999; 38: 1944-1949Crossref PubMed Scopus (63) Google Scholar, 13Goldbeck R.A. Thomas Y.G. Chen E. Esquerra R.M. Kliger D.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2782-2787Crossref PubMed Scopus (83) Google Scholar) horse heart cytochrome c. His18 and Met80are the axial heme ligands in native cytochrome c in both oxidation states. In unfolded cytochrome c, the proximal histidine (His18) remains an axial ligand by virtue of its proximity to Cys15 and Cys17, which form the thioether linkages to the porphyrin ring (14Moore G.R. Pettigrew G.W. Cytochromes c: Evolutionary, Structural and Physicochemical Aspects..in: Rich A. Springer Series in Molecular Biology. Springer-Verlag, Berlin1990Google Scholar, 15Adams P.A. Baldwin D.A. Marques H.M. Scott R.A. Mauk A.G. Cytochrome c—A Multidisciplinary Approach. University Science Books, Sausalito, CA1996: 635-692Google Scholar, 16Kranz, R., Lill, R., Goldman, B., Bonnard, G., Merchant, S., Mol. Microbiol., 29, 1998, 383, 396.Google Scholar). The sixth coordination site can either be vacant or occupied to various degrees by His33, which is the predominant non-native heme iron ligand (17Colón W. Wakem L.P. Sherman F. Roder H. Biochemistry. 1997; 36: 12535-12541Crossref PubMed Scopus (179) Google Scholar), His26 or the N-terminal amino group (18Hammack B. Godbole S. Bowler B.E. J. Mol. Biol. 1998; 275: 719-724Crossref PubMed Scopus (68) Google Scholar). During in vitro folding and refolding, cytochromec molecules can be trapped transiently in intermediate structures with various His-His ligation (9Yeh S.R. Takahashi S. Fan B. Roussseau D.L. Nat. Struct. Biol. 1997; 4: 51-56Crossref PubMed Scopus (134) Google Scholar, 11Yeh S.R. Rousseau D.L. J. Biol. Chem. 1999; 274: 17853-17859Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 19Yeh S.R. Rousseau D.L. Nat. Struct. Biol. 1998; 5: 222-228Crossref PubMed Scopus (99) Google Scholar). Structural changes induced by changes in the redox state of a heme group in a protein play a central role in channeling redox energy into conformational energy in biology. For cytochromecd 1 nitrite reductase, the factors that drive the switch between His-His and His-Met coordination of thec-type heme center in the enzyme (Fig.1) have not been elucidated. An open question is whether ligand switching at the c heme is a property of the c heme domain or of the wholecd 1 molecule. To understand the molecular details of this process, we present here the characterization of the heme ligation in the isolated c domain of the P. pantotrophus cytochrome cd 1 nitrite reductase enzyme in both oxidation states in solution. The isolated c domain consisting of the first 133 amino acid residues of the mature P. pantotrophus cytochromecd 1 nitrite reductase was expressed inEscherichia coli, as described elsewhere. 1E. Gordon, E. Steensma, and S. J. Ferguson, manuscript in preparation. Six or eight-liter cultures of E. coli in LB medium were grown overnight at 37 °C and centrifuged at 7000 × g for 15 min at 4 °C. The protein was removed from the periplasm using an osmotic shock procedure. The cell pellet was resuspended in a 0.4-culture volume of 30 mm Tris-HCl, pH 8, 20% sucrose, 1 mm EDTA, incubated 5–10 min at room temperature, and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was removed, and the pellet was resuspended in a 0.4-culture volume of ice-cold 5 mm MgSO4, stirred on ice for 10 min, and centrifuged at 10,000 ×g at 4 °C. The red-colored supernatant was loaded onto a fast-flow Q-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated using 50 mm Tris-HCl, pH 8, and 50 mm NaCl. The c domain was isolated in the reduced form. To ensure full reduction or oxidation, a small excess of ascorbic acid or ferricyanide in the solid form was added to the purified protein immediately before it was put on a Novarose PrePac S.E.-100/17 gel-filtration column (Inovata, Bromma, Sweden), which was equilibrated with 50 mm sodium phosphate buffer and 100 mmNaCl, pH 7. The protein was either directly frozen in liquid nitrogen or concentrated to 1–2 mm using 3K Centriprep (Millipore, Bedford, MA) and 3K Microsep concentrators (Filtron Technology Corp., Northborough, MA), subsequently lyophilized, and stored at −20 °C before use. The purity of c domain samples was checked by native and SDS-PAGE2 on a PhastSystem (Amersham Pharmacia Biotech, Uppsala, Sweden) and silver-stained. Care was taken to minimize proteolytic cleavage of thec domain samples by purifying and concentrating the protein at 4 °C as quickly as possible and by freezing the samples in liquid nitrogen between handling steps. The mass of the isolated c domain was determined by MALDI-TOF spectrometry. N-terminal analysis was performed to check whether the periplasmic leader sequence was cleaved off at the correct amino acid position. Equilibrium sedimentation studies were performed on 10 mmoxidized c domain samples in 100 mm sodium phosphate buffer, pH 7, 300 K, using a Centriscan 75 ultracentrifuge. The absorbance at 280 nm was recorded. UV-visible absorbance spectra between 250 and 750 nm were recorded on a Hewlett-Packard 8453 diode array spectrophotometer. Kinetic experiments to follow the ascorbate reduction of the isolatedc domain were performed using an RX2000 stopped-flow cell (Applied Photophysics, Leatherhead, UK) coupled to the HP 8453 spectrophotometer. The reaction and storage chambers of the stopped-flow cell were kept at 4.5 °C. In the experiments, 2.5 μm oxidized c domain in 50 mmphosphate buffer, pH 7, was used. Pseudo first order rate constants were determined from exponential curves of the absorbance at 417 nmversus time at 5, 10, 20, 30, 40, and 50 mmascorbate in 50 mm phosphate buffer, pH 7. From these, the second order rate constant for ascorbate reduction of the isolatedc domain was determined. Unfolding of the oxidized (16–22 μm) and reduced (8–10 μm) isolated c domain was monitored by absorbance spectroscopy as a function of the sample temperature in 50 mm sodium phosphate and 100 mm NaCl, pH 7. Thermal denaturation curves were measured between 277 and 340 K for oxidized and between 316 and 368 K for reduced protein samples, respectively. The isolated c domain was reduced with solid dithionite (end concentration 10 mm) inside an anaerobic glove box and kept overnight in the glove box before it was pipetted in an airtight cuvette and sealed. The sample temperature was measured with a thin NiCr-NiAl thermocouple (Testo 925, Göteborgs Termometerfabrik, Göteborg, Sweden), which was in direct contact with the protein solution inside the sealed cuvette. The precision of the temperature reading was within ±0.1 °C. The heating rate was manually controlled and varied between 0.25 and 1 °C per minute to ensure that thermal unfolding of the protein was at equilibrium,i.e. a change in heating rate within this regime did not result in a change of the T m. Thermal unfolding curves were measured at different wavelengths and corrected with the absorbance measured at 900 nm, a wavelength at which no protein absorbance is expected. The corrected unfolding curves were normalized and subsequently analyzed according to a two-state mechanism of unfolding (21Pace C.N. Shirley B.A. Thomson J.A. Creighton T.E. Protein Structure: A Practical Approach. IRL press, Oxford1989: 311-330Google Scholar, 22Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure: A Practical Approach. 2nd. Ed. IRL press, Oxford1997: 299-321Google Scholar) and assuming a linear dependence of the pre- and post-unfolding baselines with the temperature. The data were fitted as described in Ref. 23van Mierlo C.P.M. van Dongen W.M.A.M. Vergeldt F. van Berkel W.J.H. Steensma E. Protein Sci. 1998; 7: 2331-2344Crossref PubMed Scopus (63) Google Scholar to obtain values for T m, except that no linear dependence of the post-unfolding baseline was assumed and that ΔC p was kept constant at an estimated value of 2513 cal.mol−1.K−1 (22Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure: A Practical Approach. 2nd. Ed. IRL press, Oxford1997: 299-321Google Scholar) in fits to the data of the isolated reduced c domain. NMR samples were prepared by dissolving lyophilized protein in either 99.9% 2H2O or 90% H2O/10% 2H2O to yield 1–2 mm protein solutions in 50 mm sodium phosphate buffer and 100 mm sodium chloride, pH* 6.7. Solutions were prepared in an anaerobic glove box, and the NMR tubes were sealed with gas-tight caps. All samples contained 0.1–0.2 mm DSS as an internal standard. The c domain has a tendency to auto-oxidize and to auto-reduce, and 100% oxidized or 100% reduced samples, as detected by NMR spectroscopy, could not be made without the addition of oxidizing and reducing agents, respectively. The phenomenon of auto-reduction has been observed for other cytochromes cat high pH (24Ubbink M. Canters G.W. Biochemistry. 1993; 32: 13893-13901Crossref PubMed Scopus (26) Google Scholar). 3M. Ubbink, personal communication. Samples were oxidized or reduced in the NMR tubes by addition of small amounts of ferricyanide or sodium ascorbate, respectively, in 50 mmsodium phosphate, 100 mm sodium chloride, pH 7, using a Hamilton syringe until subsequent additions did not result in changes in the NMR spectrum. Before and after each two-dimensional1H NMR experiment, one-dimensional 1H NMR spectra of the NMR sample were recorded and compared to ensure that the sample had not deteriorated during the NMR experiment. Furthermore, an aliquot of each c domain sample was taken before and after each series of NMR experiments (lasting for several days) and checked with SDS-PAGE to see whether degradation had occurred during acquisition of the NMR experiments. All 1H NMR spectra were recorded on a Bruker DRX 500 spectrometer either at 500.13 or at 500.03 MHz. One- and two-dimensional experiments on isolated oxidized and reducedc domain were performed over the range of 278–308 K. One-dimensional 1H NMR experiments were recorded with a spectral width of 95 ppm. In two-dimensional 1H NMR experiments, the spectral width in both dimensions was 17 or 18 ppm when information on the diamagnetic region of spectra of the isolatedc domain was to be obtained. To obtain information on hyperfine shifted resonances of the isolated oxidized cdomain, a spectral width of 95 ppm was used in two-dimensional1H NMR experiments. Routinely, 2048 complex points were acquired in the direct dimension, whereas 256 complex points were recorded in the indirect dimension of two-dimensional 1H NMR experiments. Quadrature detection in the indirect dimension was accomplished using the time proportional phase incrementation method. Presaturation of the water signal was always employed during the relaxation period. Ordinary 30- and 150-ms NOESY, 31-ms clean-TOCSY (25Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1189) Google Scholar), using an mlev17 (26Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) sequence, and DQF-COSY experiments were recorded on the diamagnetic regions of spectra of the isolated c domain in both oxidation states. The relaxation delay was between 1 and 2 s. T1 inversion recovery experiments were performed on the oxidized protein in 100% 2H2O at 278, 295, and 298 K and in 90% H2O/10% 2H2O at 278 K using a relaxation delay of 5 s between individual free induction decays. Peak volumes of resolved peaks were plotted with respect to recovery delay time, and T1 values were extracted from exponential fits to the data using the XWINNMR package unless stated otherwise. In DEFT-NOESY and DEFT-TOCSY experiments (27Bertini I. Banci L. Luchinat C. Methods Enzymol. 1989; 177: 246-263Crossref PubMed Scopus (20) Google Scholar, 28Kolczak U. Han C. Sylvia L.A. La Mar G.N. J. Am. Chem. Soc. 1997; 119: 12643-12654Crossref Scopus (15) Google Scholar), the first 90° pulse in the ordinary NOESY and TOCSY pulse sequences was replaced with the modified DEFT sequence (90°-τ-180°-τ-90°) (29Becker E.D. Ferretti J.A. Farrar T.C. J. Am. Chem. Soc. 1969; 91: 7784-7785Crossref PubMed Scopus (151) Google Scholar) with τ = 100 ms. In two-dimensional DEFT 1H NMR experiments, the relaxation delay was set to 200 ms. A 5-ms DEFT-TOCSY1H NMR experiment was recorded on the isolated oxidizedc domain. NOE mixing times of 5 and 15 ms were used in DEFT-NOESY 1H NMR experiments. Two-dimensional exchange spectroscopy (EXSY) 1H NMR experiments (30Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4805) Google Scholar, 31Boyd J. Moore G.R. Williams G. J. Magn. Reson. 1984; 58: 511-516Google Scholar) were performed on samples containing mixtures of the oxidized and reduced c domain in2H2O (278 and 298 K) and in 90% H2O/10% 2H2O (278 K). Under the experimental conditions used, all 1H resonances are broadened to some extent due to intermediate exchange between oxidized and reduced molecules. NOE mixing times of 5 and 15 ms were used in the DEFT-NOESY pulse sequences to ensure that even cross peaks arising from resonances with the shortest T1 values could be observed. Two-dimensional 1H NMR data were processed using the XWINNMR package. One-dimensional and two-dimensional 1H NMR data sets were apodized using an exponential decay in the direct dimension and using a 30–40° shifted sine-bell-squared window function in the indirect dimension. After phase correction, the spectra were baseline corrected in both dimensions. Two-dimensional 1H NMR spectra were analyzed using the program XEASY (ETH Zurich, Switzerland (32Bartels C. Xia T. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 5: 1-10Crossref PubMed Scopus (1585) Google Scholar)). One-dimensional1H NMR spectra were processed and analyzed using the program Gifa (33Pons J.L. Malliavin T.E. Delsuc M.A. J. Biomol. NMR. 1996; 8: 445-452Crossref PubMed Scopus (228) Google Scholar). 1H chemical shifts were referenced using internal DSS as a standard. The isolated c domain is expressed in the reduced form in the periplasm of E. coli 1 and can be purified in the reduced state. At pH 8, the isolatedc domain remains reduced for weeks under aerobic conditions at 4 °C, as determined by absorbance spectroscopy. The absorbance spectrum of the reduced c domain (gray curve in Fig. 2 A) shows characteristic maxima at 418 nm (Soret band), 522 nm (β band), and 548 nm and 554 nm (split α bands), which are indicative of a low-spin six-coordinated heme iron. The oxidized c domain has absorption maxima at 410 and 527 nm (black curve in Fig. 2 A). Furthermore, at high protein concentrations (millimolar) a small band is observed at 696 nm (see inset in Fig. 2 A) indicative of methionine ligation to the heme (14Moore G.R. Pettigrew G.W. Cytochromes c: Evolutionary, Structural and Physicochemical Aspects..in: Rich A. Springer Series in Molecular Biology. Springer-Verlag, Berlin1990Google Scholar). For comparison, the absorbance spectra of reduced and oxidized cytochromecd 1 nitrite reductase are shown in Fig.2 C. The c domain appeared as a monomer on native gels, and mass/charge MALDI-TOF spectra did not show any indication of dimers. Sedimentation equilibrium studies with 10 μm protein solution confirmed that the isolated c domain is monomeric in solution. Although absorbance spectra of reduced c domain samples did not change over a period of weeks when stored at 4 °C, SDS-PAGE shows that the protein underwent a degradation over such periods of time (data not shown). N-terminal amino acid sequencing of an agedc domain sample, which was stored in the reduced state for a month at 4 °C, gave the sequence APEGVSALSD, showing that the first 41 residues of the c domain had been cleaved off. Mass spectrometric analysis gave a mass of 10,422 Da, confirming that most of the aged protein consisted of residues 42–133 of the cdomain sequence and that no full-length c domain was present anymore. The isolated c domain can reversibly be oxidized by ferricyanide and re-reduced by dithionite or ascorbate. The oxidizedc domain is readily reduced by ascorbate, with a second order rate constant of 20.9 ± 1.4m−1.s−1 in 50 mmphosphate buffer, pH 7.0, 4.5 °C. This is in contrast to the oxidized cd 1 nitrite reductase enzyme, which can only be reduced slowly by ascorbate (34Koppenhöfer A. Little R.H. Lowe D.J. Ferguson S.J. Watmough N.J. Biochemistry. 2000; 39: 4028-4036Crossref PubMed Scopus (35) Google Scholar). 4T. Sjögren, personal communication. The one-dimensional 1H NMR spectrum of the reduced c domain in2H2O (green curve, Fig.3 A) and H2O (black curve, Fig. 3 A) shows several outstanding features typical for c-type cytochromes. The resonances of the reduced protein exhibit a broad chemical shift dispersion brought about by the presence of the aromatic heme group (Fig. 3 A). The four downfield resonances between 9 and 10.5 ppm in the2H2O spectrum arise from the four meso protons (numbered 5, 10, 15, and 20in Fig. 4), which reside within the heme plane. Many resonances between ∼1 and −3 ppm arise from protons of the axial ligands and from other protons located close to the center of and perpendicular to the heme plane. Using two-dimensional NOESY, TOCSY, and COSY 1H NMR spectra, most of the heme proton were assigned (TableI). 5The NMR data on the isolated c domain in the reduced and oxidized state are deposited in the BioMagResBank web site and have accession numbers 4800 and 4801, respectively. Figure 4NOEs observed in two-dimensional1H NMR spectra of the isolated reduced cdomain are indicated as red lines in the crystal structure of the reduced P. pantotrophus cytochromecd 1 nitrite reductase (Protein Data Bank file 1AOF; hydrogens were generated using the program InsightII (Biosym/MSI, San Diego, CA). The c heme is also shown to the right with the four mesoprotons numbered according to the IUPAC recommendations 1999 (59Giles P.M. Pure Appl. Chem. 1999; 71: 587-643Crossref Scopus (42) Google Scholar). The c heme is covalently attached to the protein via the two sulfur atoms of Cys65 and Cys68, which are coloredyellow. The figures were drawn using a modified version of MOLSCRIPT (61Esnouf R.M. J. Mol. Graph. Model. 1997; 15: 132-134Crossref PubMed Scopus (1793) Google Scholar) and were rendered using Raster3D (20Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 673-869Crossref Scopus (2855) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IChemical shifts of the 1H resonances of the heme protons and of the side-chain protons of the two axial ligands, His69 and Met106, and of Trp109 of the P. pantotrophus-isolated c domain in the reduced and oxidized state in 50 mm sodium phosphate and 100 mm sodium chloride in 100% 2H2O, pH* 6.7Atom 1-aHeme atom nomenclature according to the IUPAC recommendations (59), and hydrogen nomenclature for amino acid residues according to the recommendations for the presentations of NMR structures of proteins (60).Heme atom name or residueδ red 1-bred, reduced; ox, oxidized.278 K ± 0.01δ red 300 K ± 0.01δ ox 278 K ± 0.05δ ox 295 K ± 0.05T1 278 Kppmms2Methyl3.523.528.799.87ND1-cND, not determined.7Methyl4.344.3238.2236.407312Methyl3.393.3814.2015.7910118Methyl3.363.3644.4942.191305Meso10.2710.266.727.30ND10Meso9.529.501.402.67ND15Meso9.369.397.958.74ND20Meso9.319.31−1.96−0.58ND2 methine5.365.36NDNDND2 methyl0.630.68NDNDND4 methine6.406.38NDNDND4 methyl2.502.49NDNDND(Hɛ)3Met106−2.51−2.54−12.23−9.914Hγ2 1-dThe higher field resonances of the two Hβ and the two Hγ protons have arbitrarily been assigned to Hβ2 and Hγ2, respectively. xcMet106−2.20−2.19NDNDNDHγ3 1-dThe higher field resonances of the two Hβ and the two Hγ protons have arbitrarily been assigned to Hβ2 and Hγ2, respectively.Met106−0.94−1.06−33.69−26.1314Hβ2 1-dThe higher field resonances of the two Hβ and the two Hγ protons have arbitrarily been assigned to Hβ2 and Hγ2, respectively.Met106−1.87−1.92−9.84−9.137Hβ3 1-dThe higher field resonances of the two Hβ and the two Hγ protons have arbitrarily been assigned to Hβ2 and Hγ2, respectively.Met1060.270.195.985.73NDHδ1His6910.47 1-eChemical shift determined in 90% H2O/10% 2H2O only.10.38 1-eChemical shift determined in 90% H2O/10% 2H2O only.12.21 1-eChemical shift determined in 90% H2O/10% 2H2O only.13.47 1-eChemical shift determined in 90% H2O/10% 2H2O only.1 1-fT1 estimate (±30%) from the null in intensity (T1 = τnull/ln2) in an inversion-recovery experiment.Hδ2His690.500.48NDNDNDHɛ1His690.570.57−12.23−11.303 1-gDetermined at 298 K.Hδ1Trp1095.815.82NDNDNDHɛ1Trp109ND6.25 1-eChemical shift determined in 90% H2O/10% 2H2O only.NDNDNDHζ2Trp1098.598.57NDNDNDHη2Trp1097.857.83NDNDNDHζ3Trp1097.337.31NDNDNDHɛ3Trp1097.307.29NDNDNDT1 values are reported for non-overlapping hyperfine shifted resonances only.1-a Heme atom nomenclature according to the IUPAC recommendations (59Giles P.M. Pure Appl. Chem. 1999; 71: 587-643Crossref Scopus (42) Google Scholar), and hydrogen nomenclature for amino acid residues according to the recommendations for the presentations of NMR structures of proteins (60Markley J.L. Bax A. Arata Y. Hilbers C.W. Kaptein R. Sykes B.D. Wright P.E. Wüthrich K. J. Mol. Biol. 1998; 280: 933-952Crossref PubMed Scopus (186) Google Scholar).1-b red, reduced; ox, oxidized.1-c ND, not determined.1-d The higher field resonances of the two Hβ and the two Hγ protons have arbitrarily been assigned to Hβ2 and Hγ2, respectively.1-e Chemical shift determined in 90% H2O/10% 2H2O only.1-f T1 estimate (±30%) from the null in intensity (T1 = τnull/ln2) in an inversion-recovery experiment.1-g Determined at 298 K. Open table in a new tab T1 values are reported for non" @default.
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• W2080290351 date "2001-02-01" @default.
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