Biochem. J. (1990) 272, 545-548 (Printed in Great Britain)

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Spectroscopic, thermodynamic and kinetic properties of Candida nitratophila nitrate reductase Christopher J. KAY,* Michael J. BARBER,*§ Larry P. SOLOMONSON,* David KAU,t Andrew C. CANNONS*t and Charles R. HIPKINt * Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa, FL 33612, U.S.A., and t Biochemistry Research Group, School of Biological Sciences, University College of Swansea, Swansea SA2 8PP, Wales, U.K.

Visible spectra of oxidized and reduced Candida nitratophila assimilatory NAD(P)H: nitrate reductase yielded absorbance maxima of 413 nm and 423 nm, and 525 nm and 555 nm respectively, characteristic of a b.-type cytochrome. E.p.r. spectra of the partially reduced enzyme revealed a single Mo(V) species (g, = 1.9957, g2 = 1.9664 and g3 = 1.9658) exhibiting superhyperfine coupling to a single proton [A('H)av = 1.4 mil. Oxidation-reduction midpoint potentials (EO) (25 °C, pH 7) for the haem and Mo-pterin prosthetic groups were determined by visible and e.p.r. potentiometric titrations and yielded values of Eo = -174 mV (n = 1) for the haem and Eo = -3 mV and Eo = -27 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Comparison of initial rates of the NADH-oxidizing and nitrate-reducing partial activities at various ionic strengths indicated electron transfer from reduced haem to Mo was rate-limiting during turnover. These results suggest a close similarity between Candida nitratophila and Chlorella vulgaris nitrate reductases.

INTRODUCTION Nitrate reductase (NR; EC 1.6.6.1) catalyses the initial and rate-limiting step, the NAD(P)H-dependent reduction of nitrate to nitrite, in the assimilation of inorganic nitrogen (Solomonson & Barber, 1990; Guerrero et al., 1981). The enzyme has been isolated from a variety of sources, ranging from algae (Howard & Solomonson, 1982) to higher plants (Fido & Notton, 1984), and shown to contain FAD, a b5-type cytochrome and Mopterin prosthetic groups in a 1:1:1 stoichiometry per subunit. The flavin and Mo-pterin centres have been identified as the binding sites for NAD(P)H and nitrate respectively, whereas the b5-type cytochrome is believed to mediate the intramolecular transfer of reducing equivalents from FAD to Mo-pterin. This hypothesis is supported by recent midpoint-potential measurements for all three prosthetic groups of Chlorella nitrate reductase (Kay et al., 1988), which showed the haem potential (Eo =- 164 mV, n = 1) to be intermediate between that of FAD (Eo= -272 mV, n =2) and Mo-pterin (E = -10 mV, n = 2). Similar values have been obtained for spinach NR (Kay et al., 1989). Assimilatory NR exhibits a number of partial activities, using artificial electron donors and acceptors (Solomonson et al., 1986). Limited proteolysis of both the Chlorella (Solomonson et al., 1986) and spinach (Notton et al., 1989) enzymes has identified the prosthetic groups essential for each partial activity. NADH: ferricyanide reductase (NADH: FR) activity requires FAD, whereas NADH: cytochrome c reductase (NADR: CR) and NADH: dichlorophenol-indophenol (NADH: DR) activities require FAD and haem. In contrast, reduced-flavin: nitrate reductase (FH2: NR) activity requires both haem and Mo-pterin, whereas reduced Methyl Viologen: nitrate reductase (MV: NR) activity requires only Mo-pterin. Recently, NR has been purified to electrophoretic homogeneity from the yeast Candida nitratophila (Hipkin et al., 1986). Several properties of the protein, including its quaternary structure

(homotetramer), subunit Mr (95 kDa) and reversible inhibition by cyanide have suggested a close similarity to the Chlorella enzyme.

MATERIALS AND METHODS Enzyme purification NR was purified from Candida nitratophila (N.Y.C.C. 556) as previously described (Hipkin et al., 1986). Enzyme concentrations were estimated by using an absorption coefficient of 117 mm-'1. cm-' at 413 nm. The Mo content was determined calorimetrically (Solomonson et al., 1975) and was typically 0.85 mol of Mo/mol of haem. Enzyme assays Initial rates of the full (NADH: NR) and partial (NADH: FR, NADH:CR, NADH: DR, FH2: NR and MV: NR) enzyme activities were measured as previously described (Kay and Barber, 1986; Barber & Notton, 1990) and are expressed in terms of ,umol of substrate consumed or product produced/min per nmol of NR haem, normalized to a value of two reducing equivalents. To ensure a full complement of FAD, which dissociates during enzyme purification (Hipkin et al., 1986), all assays were performed in the presence of 5 tM-FAD and 0.1 mM-EDTA. Potentiometry Visible and room-temperature e.p.r. potentiometric titrations were performed as described by Dutton (1978) and Kay & Barber (1990). Enzyme was reduced with MV+ (20mM) or oxidized with K3Fe(CN)6 (20 mM) in the presence of the following mediators (2-20 /SM each): 2,6-dichlorophenol-indophenol (Eo = + 217 mV), 1,2,-naphthoquinone (Eo = + 135 mV), 1,4-naphthoquinone (Eo = + 60 mV), Methylene Blue (Eo + 10 mV), 2,5-dihydroxybenzoquinone (Eo = -60 mV), 2-hydroxy- 1,4naphthoquinone (Eo = -137 mV), anthraquinone-2,7-disulphonate (Eo = 182 mV) and anthraquinone-2-sulphonate (Eo = =

-

Abbreviations used: NR, nitrate reductase; NADH: NR, NADH: nitrate reductase; NADH: FR, NADH: ferricyanide reductase; NADH: CR, NADH: cytochrome c reductase; NADH: DR, NADH: dichlorophenol-indophenol reductase; FH2: NR, reduced-flavin: nitrate reductase; MV: NR, reduced Methyl Viologen: nitrate reductase; MV+, reduced Methyl Viologen radical cation; EO, oxidation-reduction midpoint potential. § To whom correspondence should be addressed.

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546 -225 mV). Eo values were determined by fitting the experimental data to the respective Nernst equation for a single (haem) or two sequential (Mo-pterin) one-electron reduction processes using a least-squares procedure. D

Spectroscopy Visible spectra were recorded by using a Shimadzu UV260 spectrophotometer, and e.p.r. spectra were obtained using a Varian E109 Century Series spectrometer operating at 9 GHz with 100 kHz modulation. Double integrations of e.p.r. spectra were performed as described by Wyard (1965) using potassium nitrosodisulphonate (ICN Pharmaceuticals) as standard. Computer simulations of experimental spectra were performed as described by Lowe (1978).

RESULTS Visible absorption spectra obtained during anaerobic reduction of C. nitratophila NR utilizing MV+ as the reductant at pH 7.0 are shown in Fig. 1. Dye mediators were omitted from the titration, since MV+ is a facile reductant for NR and some mediators absorb strongly in the visible region of the spectrum. The absorbance changes were primarily due to reduction of the haem prosthetic group, since FAD and Mo-pterin are comparatively weak chromophores in the visible region. Reduction of the haem resulted in a shift in the Soret peak from 413 nm in the oxidized enzyme to 423 nm in the reduced enzyme, with an isosbestic point at 416 nm. Absorption changes during reduction were also observed in the a- (555 nm) and ,8- (525) regions of the spectrum. The results of visible potentiometric titrations of the haem prosthetic group are shown in Fig. 2. Haem reduction or oxidation, monitored at 423 nm, conformed to a reversible, n = 1 redox process with a midpoint potential of -174 mV. The low-temperature Mo(V) e.p.r. spectrum of partially reduced NR, shown in Fig. 3, exhibited near axial symmetry and showed superhyperfine splitting due to interaction with a single, s = 1/2, nucleus. Computer simulation of the experimental spectrum indicated g-values of g, = 1.9957,g2= 1.9664 and g3 = 1.9658, and superhyperfine coupling constants of A, = 1.0 mT, A2= 1.1 mT and A3 =1.4mT respectively. Double integration

x

0

Eo (mV against standard hydrogen electrode)

Fig. 2. Behaviour of the haem during potentiometric titration of NR NR (0.9 #M-haem) in 50 mM-Mops buffer, pH 7.0, in the presence of dye mediators (2,M each mediator), was reduced using MV+20 mM) or oxidized using Fe(CN)63- (20 mM). Haem reduction, after attainment of equilibrium, was monitored at 423 nm using 416 nm as an isosbestic wavelength. Data points, obtained in both reductive (0) and oxidative (-) directions, were fitted to a reversible n = 1 redox process with an Eo value of -174 mV.

Magnetic field (mT) Fig. 3. Mo(V) e.p.r. spectra of NR (a) NR (11 uM-haem) in 50 mM-Mops buffer, pH 7.0, was poised at 0 mV in the presence of dye mediators (20 /ZM each mediator) and rapidly frozen in liquid N2. The e.p.r. spectrum was recorded at 173 K using a microwave power of 10 mW and a modulation amplitude of 0.32 mT. (b) Computer simulation of the experimental spectrum shown in (a), using the following parameters: g, = 1.9957, 92 = 1.9664, g3 = 1.9658, A(1H)1 = 1.3 mT, A(1H)2 = 1.27 mT and A(1H)3 = 1.50 mT. The field scale corresponds to a frequency of 8.989 GHz. .0 Co

350

400

450

550 500 Wavelength (nm)

600

65

Fig. 1. Spectral changes accompanying reduction of NR NR (0.9 gM-haem) in 50 mM-Mops buffer, pH 7.0, was reduced ), anaerobically with MV+ . The spectra correspond to oxidized ( 72% reduced (----), 89% reduced (---) and 100 % reduced

(. .)-

of the e.p.r. signal indicated conversion of approx. 39 % of the Mo into Mo(V). The behaviour of the Mo(V) e.p.r. signal during roomtemperature potentiometric titrations is shown in Fig. 4. The room-temperature e.p.r. spectrum was similar in overall lineshape to that observed for frozen samples, but substantially broader and less well resolved. At potentials above + 100 mV, the Mo(V) e.p.r. signal was undetected. However, as the potential was decreased, the Mo(V) signal increased, reaching a maximum

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54:7

E 0.50 C

.D 0.401 0.30 .0~~~~~~~~

100 -150 -100 -50 0 50 Eo (mV against standard hydrogen electrode)

Fig. 4. Behaviour of the Mo(V) e.p.r. spectrum during room-temperature potentiometric titration of NR NR (11 /M-haem) in 50 mM-Mops buffer, pH 7.0, in the presence of dye mediators (20 /M each mediator) was reduced using MV+(20 mM) or oxidized using Fe(CN)63-. Mo(V) e.p.r. spectra were recorded after attainment of equilibrium using 50 mW microwave power and 0.32 mT modulation amplitude, and the integrated signal intensity, obtained in both reductive (0) and oxidative (0) directions, was plotted against the applied potential. The curve fitted to the data points corresponds to two coupled one-electron redox processes with Eo values of -3 mV and -27 mV.

amplitude at approx. -15 mV, and then decreased, becoming undetectable at potentials lower than - 150 mV. Integration of the maximum signal intensity indicated conversion of 46% of the enzyme-bound Mo into Mo(V). The lineshape of the Mo(V) signal was unchanged throughout the course of the titration, which was fully reversible. This behaviour was typical of a process involving two coupled one-electron reductions corresponding to the reactions: E,

E2

Mo(VI) - Mo(V) -

Mo(IV)

where E, and E2 represent the midpoint potentials for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Midpoint potentials obtained for these couples at pH 7 corresponded to -3 mV and -27 mV respectively. Maximal rates for the full and partial enzyme activities catalysed by Candida NR were determined under conditions of constant pH and ionic strength. Maximum NADH: NR activity corresponded to 9.3 ,umol of 2e/min per nmol of haem [I (ionic strength) 0. 1, pH 7.0]. Decreasing I to 0.02 resulted in an approx. 40 % decrease in activity (5.9 ,umol of 2e/min per nmol of haem). In contrast, NADH: FR and NADH: CR activities were unaffected by the value of Iand exhibited rates of 56 and 33 ,smol of 2e/min per nmol of haem respectively (pH 7, I0.1). Examination of the nitrate-reducing activities showed that FH2: NR activity was stimulated by elevated I values (l1.5,umol of 2e/min per nmol of haem, I0.1; 5.7 #smol of 2e/min per nmol of haem, I0.02), whereas MV: NR activity remained relatively constant (25.9,umol of 2e/min per nmol of haem, I0.1).

DISCUSSION The preceding results provide the first detailed examination of the spectroscopic and thermodynamic properties of the cytochrome and Mo-pterin prosthetic groups of a yeast NR. Absorbance maxima obtained for both the oxidized and reduced enzyme were dominated by the haem and were identical with values published for both the Chlorella and spinach enzymes (Kay et al., 1988; Fido et al., 1979). The redox potential obtained for the haem centre (Eo = - 17'4 mV) was very similar to that of

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the Chlorella enzyme (E = -162 mV) (Kay et al., 1988) and approx. 50 mV lower than the potential of the haem in spinach NR (Eo = - 120 mV) (Kay et al., 1989). Although these results suggest only minor changes in the haem environment of Candida, Chlorella and spinach NR, they can be contrasted with the more positive haem potential reported for Monoraphidium braunii NR (Eo =-74 mV) (De La Rosa et al., 1980). The close similarity in the structure of the Mo centre between Candida, Chlorella and spinach NRs was evident from the similarity in their Mo(V) e.p.r. parameters (Candida: g-V= 1.977, AlHav = 1. 17mT; Chlorella: gav = 1.977, AlHav = 1.35 mT; spinach: gy. = 1.977, AlHav = 1.30 mT) (Kay et al., 1989; Gutteridge et al., 1983) and the Mo(VI)/Mo(V) and Mo(V)/ Mo(IV) midpoint potentials, which were clustered around average values of -2 mV and -32 mV respectively (Kay et al., 1988, 1989). Initial-rate studies of Chlorella and spinach NRs have shown differences in their response to changes in ionic strength. For Chlorella NR, increased I enhanced both FH2: NR and NADH: NR activities by increasing the rate of electron transfer from haem to Mo-pterin. This has been interpreted as haem -. Mo electron transfer comprising the rate-limiting step in catalysis by Chlorella NR (Kay & Barber, 1986). In contrast, all spinach NR partial activities were higher than the NADH: NR activity, suggesting no partial activity contained the rate-limiting step (Barber & Notton, 1990). For Candida NR, increased Istimulated FH2: NR and NADH: NR activities, analogous to the Chlorella results, suggesting haem -. Mo rate-limitation. The diaphorase partial activities for Candida NR were lower than those for Chlorella NR, suggesting that structural differences between the two nucleotide-binding sites that allow Candida, but not Chlorella, to utilize NADPH, may result in differing specific activities for these partial activities. In summary, Candida NR compares more closely with the corresponding Chlorella enzyme than that of spinach in terms of its subunit composition, haem and Mo E, value and ratelimitation by haem-Mo intramolecular electron transfer. However, it is apparent that assimilatory NRs obtained from diverse sources exhibit very similar structural and mechanistic features. This work was supported by grants GM 32696 from the National Institutes of Health and GAM 88-37120-3871 from the U.S. Department of Agriculture.

REFERENCES Barber, M.J. & Notton, B.A. (1990) Plant Physiol. 93, 537-541 De La Rosa, M.A., Diez, M.A., Vega, J.M. & Losada, M. (1980) Eur. J. Biochem. 106, 249-256 Dutton, P.L. (1978) Methods Enzymol. 54, 411-435 Fido, R.J. & Notton, B.A. (1984) Plant Sci. Lett. 37, 87-91 Fido, R.J-., Hewitt, E.J., Notton, B.A., Jones, O.T.G. & NasrulhaqBoyce, A. (1979) FEBS Lett. 99, 180-182 Guerrero, M.G., Vega, J.M. & Losada, M. (1981) Annu. Rev. Plant Physiol. 32, 169-204 Gutteridge, S., Bray, R.C., Notton, B.A., Fido, R.J. & Hewitt, E.J. (1983) Biochem. J. 213, 137-142 Hipkin, C.R., Ali, A.H. & Cannons, A.C. (1986) J. Gen. Microbiol. 132,

1997-2003 Howard, W.D. & Solomohson, L.P. (1982) J. Biol. Chem. 257, 1024310250 Kay, C.J. & Barber, M.J. (1986) J. Biol. Chem. 261, 14125-14129 Kay, C.J. & Barber, M.J. (1990) Anal. Biochem. 184, 11-15 Kay, C.J., Barber, M.J. & Solomonson, L.P. (1988) Biochemistry 27, 6142A-149 Kay, C.J., Barber, M.J., Notton, B.A. & Solomonson, L.P. (1989) Biochem. J. 263, 285-287

548 Lowe, D.J. (1978) Biochem. J. 171, 649-651 Notton, B.A., Fido, R.J., Whitford, P.N. & Barber, M.J. (1989) Phytochemistry 28, 2261-2266 Solomonson, L.P. & Barber, M.J. (1990) Annu. Rev. Plant Physiol. Mol. Biol. 41, 225-253

C. J. Kay and others Solomonson, L.P., Lorimer, G.H., Hall, R.L., Borscher, R. & LeggettBailey, J. (1975) J. Biol. Chem. 250, 4120-4127 Solomonson, L.P., Barber, M.J., Robbins, A.P. & Oaks, A. (1986) J. Biol. Chem. 261, 11290-11294 Wyard, S.J. (1965) J. Sci. Instrum. 42, 768-769

Received 7 August 1990/1 October 1990; accepted 8 October 1990

1990