Dependence of the Proton Magnetic Resonance Spectra on The

Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part I, pp. 3292-3295, December 1973 Dependence of the Proton Magnetic Resonance Spectra on the Oxidation State of Flavodoxin from Clostridium MP and from Peptostreptococcus elsdenii (natural spin label/paramagnetic line broadening/peak assignments/species dependence/conformation) THOMAS L. JAMES*, MARTHA L. LUDWIGt, AND MILDRED COHN* * Department of Biophysics and Physical Biochemistry, School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania 19174; and t Biophysics Research Division, Institute of Science and Technology, University of Michigan, Ann Arbor, Mich. 48105 Contributed by Mildred Cohn, July 25, 1973 ABSTRACT The broadening of protein nuclear mag- In this paper, the proton nuclear magnetic resonance netic resonances in the spectra of the semiquinone forms (NMR) spectra of flavodoxins from P. elsdenii and Cl. MP in of flavodoxins derived from Clostridium MP and Pepto- streptococcus elsdenii relative to the resonances in the each of the three oxidation states were compared. The phe- oxidized and reduced forms is highly selective. Spectra nomena observable in the present investigation differ from from both species of flavodoxin indicate that conforma- previous NMR investigations of iron-containing electron tional differences between the oxidized and fully reduced carriers (16, 17) in two essential ways: (1) three oxidation states are minor and, consequently, the broadening in the semiquinone form is ascribed to the paramagnetic effect states of flavodoxins are available for comparison, two dia- of the flavin free radical. The chemical shifts of the paramagnetic species, the oxidized and fully reduced forms, and magnetically broadened lines are used in conjunction with one paramagnetic species, the semiquinone form; (2) the x-ray crystallographic models to assign peaks to amino- dominant effect of the unpaired electron in the flavodoxin acid residues in the proximity of the flavin mononucleo- is manifested in the nuclear relaxation as moni- tide. Species-dependent differences in the spectra can semiquinone generally be attributed to differences in amino-acid com- tored by linewidths rather than contact shifts since its elec- position and sequence. The spectra from both species of tron spin relaxation time, unlike that of iron, is very long; a flavodoxin indicate that there is slow exchange between value of the order of 10-8 sec can be estimated from the elec- oxidized and semiquinone forms or reduced and semi- tron spin resonance linewidth (18). The flavin free radical quinone forms of the flavodoxins with a limit of kex < 50 on sec'I for the exchange rate. selectively broadens the resonances of protons amino acids located near the isoalloxazine ring, permitting, in principle, Flavodoxins are small flavoproteins (molecular weight mapping of amino acids in the vicinity of the active site. The 15,000-22,000) that replace ferredoxins as low-potential elec- semiquinone form of flavodoxin thus provides a natural site- tron carriers. Several organisms, including anaerobes (1-5), specific probe of the active-site environment. photosynthetic bacteria (6), blue-green (7, 8) and eukaryotic Recently, paramagnetic nitroxide radicals (spin labels) have algae (9), nitrogen-fixing aerobes (10, 11), and Escherichia coli been attached at specific sites on enzymes to perturb the (12), synthesize flavodoxins. The purified proteins all contain proton NMR spectra by broadening nuclear resonances on one flavin mononucleotide per molecule, with no other pros- those amino-acid residues near the unpaired electron of the thetic groups or bound metal ions. radical (19-21). For flavodoxins, we have a natural spin label Flavodoxins do not react directly with pyridine nucleotides probe of the active-site environment without the possible or other oxidizable substrates but rather act as substrates for complications arising from introduction of an extrinsic bulky other proteins. The oxidation-reduction potential for the one- group. Unlike the nitroxide free radical, however, the fiavin electron reduction of the semiquinone radical form is free radical suffers from electron delocalization (22), which in- about -0.4 V (13), in the range typical for ferredoxins. In terferes with absolute quantitation of distances. flavodoxins derived from Peptostreptococcus elsdenii and Amino-acid residues near the active site whose geometry is Clostridium MP, the potentials of the two one-electron steps conserved in both flavodoxins should be discernible by NMR. are separated by more than 0.2 V (5, 13) so that the oxi- Assignment of resonances to these residues may be made in dized, semiquinone, and fully reduced forms of the protein are conjunction with the x-ray data on Cl. MP flavodoxin. Com- each accessible for chemical and structural studies. Re- parison of the proton NMR spectra of the diamagnetic oxi- cent x-ray studies have shown that the three-dimensional dized and completely reduced forms also provides a sensitive structures of flavodoxins from Desulfovibrio vulgaris (14) and means of monitoring any conformational changes that may Cl. MP (15) are very similar in spite of the differences in com- occur on reduction. In addition, the NMR measurements position and the greater chain length (about 10 residues) of yield information concerning the rate of exchange between the D. vulgaris protein. In both structures the isoalloxazine the oxidized or fully reduced flavodoxins and the semiquinone ring is bound near the surface, but the flavin-protein interac- form. tions differ somewhat in the two species. EXPERIMENTAL The flavodoxins from C1.MP and P. elsdenii were prepared as Abbreviations: DSS, sodium 2,2-dimethyl-2-silapentanesulfo- described (3, 5). After lyophilization of solutions in phosphate nate; NMR, nuclear magnetic resonance. buffer (pH 7.5), the samples were dissolved in 99.97% deu- 3292 Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Proton NMR of Flavodoxins 3293 terium oxide, allowed to stand from 4 to 16 hr at room tem- perature to permit proton-deuterium exchange, lyophilized, and redissolved in deuterium oxide. This procedure was re- peated three times. The fully reduced form of flavodoxin was obtained by addition of an excess of the required 1 mol of sodium dithionite per mol of flavodoxin under a nitrogen atmosphere. The violet semiquinone form was quickly gen- Reduced ,, erated from the pale yellow reduced form by injecting small volumes of air through a septum seal on the NMR tube. Reoxidation of the semiquinone form is slow (13) and required that the sample be spun for about 1 hr after exposure to air. The NMR spectra of the initial oxidized samples and of the reoxidized samples were identical. Semiquinone A Varian HA-220 spectrometer equipped with a pulse unit and computer was used to obtain continuous wave spectra Odi with time-averaging and Fourier transform spectra at 220 MHz and 190. Chemical shifts were measured relative to the methyl resonance of external sodium 2,2-dimethyl-2-silapen- Oxidized tanesulfonate (DSS). I RESULTS AND DISCUSSION The well-resolved proton NMR spectra of P. elsdenii flavo- 9 8 7 6 5 4 3 2 1 0 -1 doxin in various oxidation states are shown in Fig. 1, and the Chemical Shift (ppm from DDS) FIG. 2. Proton NMR spectra of 4.5 mM flavodoxin (Cl. MP) in potassium phosphate buffer (pD 7.5) in three different oxidation states. Each spectrum shown is an average of 10 scans. spectra of Cl. MP flavodoxin in three oxidation states are presented in Fig. 2. Many of the differences between the spectra of the oxidized forms of the two flavodoxin species F Reduced 1i II (Figs. 1A and 2A) can be attributed to differences in amino- acid composition. For example, the alanine peak at 1.3-1.4 in with I I ppm is much larger in the P. elsdenii spectrum, accord the presence of 18 Ala (23) rather than the 6 Ala (24) found I'1 in Cl. MP flavodoxin. The aromatic region appears less well- E. -,-50% Semiquinone + - 50% Reduced resolved in the Cl.MP spectrum, partly as a result of one addi- Ai tional Tyr and one additional Phe in the molecule. Differences between the spectra shown here and that of oxidized Cl. pasteurianum flavodoxin (25) also reflect alterations in amino- I acid composition (1, 26). Spectra of the oxidized and reduced forms of P. elsdenii flavodoxin are nearly identical (Fig. 1A and F). The peak at -0.81 ppm in the spectrum of the oxidized form is shifted by C Sermquinone 0.07 ppm downfield in the spectrum of the reduced form. There is some possibility that the other perturbed peaks are shifted by 0.01-0.02 ppm, but that cannot be stated with certainty. The small shift of the -0.81 ppm peak is probably caused by a small change in the electron density of the flavin Ii ring in going from the oxidized to the reduced form, such that the ring current effect on the chemical shift is slightly at- A. Oxidized 1J4 tenuated in the reduced flavin relative to the oxidized flavin. It is also conceivable that a slight change in the orientation of the flavin ring may occur between the oxidized and reduced 10 9 8 7 6 5 4 3 2 0 -I species, in addition to the electron density alteration. Chemical Shift (ppm from DSS) The spectra of the oxidized and fully reduced forms of Cl. FIG. 1. Proton NMR spectra of 4.8 mM flavodoxin (P. MP flavodoxin are virtually indistinguishable (Fig. 2A and elsdenii) in potassium phosphate buffer (pD 7.5) under various C). Thus, the spectral data imply that there can be no major conditions of oxidation or reduction. In each case the Fourier conformational differences between the oxidized and reduced transform NMR spectrum is shown for the sum of 3000 transients contrast to with an- with a 0.2-sec sampling time.

Load more