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Fig. 2. Oxidation of BFLt-containing (as isolated) neuronal deoxyferrous NOS at -30° in the presence of 1 mM NHA. Shown are the spectra of dithionite-reduced BH4-containing NOS (dotted line), as well as the spectra recorded immediately (within 2 min, continuous line) and 64 min (dashed line) after O2 addition. Inset: The visible part of the spectra at greater magnification and vertically offset for clarity. Experimental conditions: 2.1 nM NOS, 1 mM NHA, 50 mm kpi (pH 7.2), 1 mM CHAPS, 0.5 mM EDTA, 1 mM 2-mercaptoethanol, 0.23 mM sodium dithionite, and 50% (v/v) ethylene glycol. See Experimental Procedures for further details.

5-methyl-BH4 mimicked the effects of BH4. The latter observation is particularly significant, because the structure of this BH4 analog, which supports NO synthesis,6 precludes reversible two-electron oxidation, while allowing one-electron oxidation. From these two studies we concluded that BH4 functions as an obligate one-electron donor to the oxyferrous complex during NO synthesis.21 The BH3' radical that is formed in the process has now been demonstrated directly by electron spin resonance spectroscopy.22-24

Comparison with Published Results

There have been several studies applying the same technique to other P-450-type enzymes. In most cases formation of an oxyferrous complex with a red-shifted absorbance maximum between 416 and 423 nm was reported,13-16'25 similar to what we found with NOS. Interestingly, with cytochrome P-450cam and P-450scc blue-shifted intermediates (A.max 405 nm) were observed that were ascribed to the oxyferryl state (FeIV=0).15'26'27 The spectrum with Amax at 404 nm that we observed immediately after addition of oxygen to BH4-containing NOS in the presence of arginine was tentatively attributed to the same species.20 Definitive evidence supporting this assignment is still lacking.

In line with our observations, Ledbetter et al. independently reported formation of an Fen02 complex, absorbing at 419 nm, using the same technique and almost identical conditions.28 Curiously, Ledbetter et al. achieved optimal complex formation in the presence of iVG-methyl-L-arginine (NMA), whereas we observed instantaneous reoxidation without any detectable intermediate in the presence of that substrate analog (our unpublished observations, 1997). The cause of these and other subtle differences remains to be determined. Interestingly, NMA stimulates 02~ formation by inducible NOS.29 A similar stimulation of the uncoupled reaction cycle could explain the rapid reoxidation we observed in the presence of NMA.

22 A. R. Hurshman, C. Krebs, D. E. Edmondson, B. H. Huynh, and M. A. Marietta, Biochemistry 38, 15689 (1999).

23 N. Bee, A. C. F. Gorren, B. Mayer, P. P. Schmidt, K. K. Andersson, and R. Lange, J. Inorg. Biochem. 81,207 (2000).

24 P. P. Schmidt, R. Lange, A. C. F. Gorren, E. R. Werner, B. Mayer, and K. K. Andersson, J. Biol. Inorg. Chem. 6, 151 (2001).

25 R. C. Tuckey and H. Kamin, J. Biol. Chem. 257,9309 (1982).

26 R. Lange, C. Larroque, and J. E. van Lier, Biochem. Life Sci. Adv. 7, 137 (1988).

27 R. Lange, G. Hui Bon Hoa, C. Larroque, and I. C. Gunsalus, in "Cytochrome P450: Biochemistry and Biophysics" (I. Schuster, ed.), p. 272. Taylor & Francis, London, 1989.

28 A. P. Ledbetter, K. McMillan, L. J. Roman, B. S. S. Masters, J. H. Dawson, and M. Sono, Biochemistry 38, 8014 (1999).

29 H. M. Abu-Soud, P. L. Feldman, P. Clark, and D. J. Stuehr, J. Biol. Chem. 269, 32318 (1994).

The oxyferrous complex of NOS has also been observed by stopped-flow/ rapid-scan optical spectroscopy.30'33 With this method the absorbance maximum appeared at significantly higher wavelengths (427-430 nm), although one group reported a maximum at 420 nm.31 This has caused some controversy regarding the true nature of the intermediates observed by us and others. In fact, most of the differences may be artifactual. Manual addition and mixing of O2, followed by scanning of the first spectrum, puts a lower limit in the range of minutes to our method. Because several of the intermediates decayed on a similar time scale (ti/2 3-8 min), the absorbance maxima observed by us represent mixtures of the initial state (deoxyferrous NOS, absorbing at 410-412 nm), the final state (ferric NOS, absorbing at 394 or 418 nm depending on the heme spin state), and the oxyferrous complex, for which we estimate an absorption maximum between 420 and 425 nm. Some variation appears to be due to the presence of different substrates and pterin analogs. Similar subtle variations in peak position have been reported for cytochrome P-450 in the presence of different substrates.25

Advantages and Drawbacks

The main general advantage of subzero optical absorption spectroscopy is that the application of low temperatures combined with the obligatory use of a cryosolvent enable detection and characterization of reaction intermediates that are inaccessible to research by other methods. On the other hand, for the same reason the physiological significance of such intermediates is not always clear. For our studies we chose a 1:1 (v/v) mixture of ethylene glycol and aqueous buffer, because this did not affect the spectra of ferric and ferrous NOS, while the enzyme remained active.20'28 Nevertheless, the substantially diminished activity and the decrease in the affinity of NOS for BH4 indicate that the cryosolvent is not entirely innocuous.

As already mentioned, NOS binds only one equivalent of BH4 per dimer in 50% ethylene glycol. Because the enzyme species with one BH4 per dimer may be significant physiologically, this property of ethylene glycol is in some respects fortunate. However, as it prevents us from studying the pterin-saturated enzyme, we are currently exploring other cryosolvents as well.

A particularly useful aspect of our experimental procedure is that it provides a convenient method to generate ferrous NOS for single-turnover studies. Reduction of the enzyme with a small excess of sodium dithionite at room temperature, and

30 H. M. Abu-Soud, R. Gachhui, F. M. Raushel, and D. J. Stuehr, J. Biol. Chem. 272, 17349 (1997).

31 H. Sato, I. Sagami, S. Daff, and T. Shimizu, Biochem. Biophys. Res. Commun. 253, 845 (1998).

32 M. Couture, D. J. Stuehr, and D. L. Rousseau, J. Biol. Chem. 275, 3201 (2000).

33 S. Boggs, L. Huang, and D. J. Stuehr, Biochemistry 39, 2332 (2000).

subsequent addition of at —30°, circumvents complications by the remaining reductant, because at that temperature reduction of NOS by sodium dithionite is extremely slow (t\n on the order of weeks; our unpublished observations, 1998). It is this simple and effective way to achieve a situation in which only the electron on the heme is available for oxygen reduction that enabled us to conclude that BH4 must serve as the donor of the second electron in the reaction cycle with arginine. Furthermore, as the sample chamber is continuously flushed with nitrogen, anaerobic conditions are guaranteed. One can apply the same experimental procedure to generate samples for other spectroscopic techniques, such as electron paramagnetic resonance (EPR) spectroscopy.23

One disadvantage of the method is that about 2 min must elapse before the first spectrum after O2 addition can be measured. It is mainly because of this limitation that, for some intermediates, spectral decomposition, and hence determination of the absorption spectra, could not be achieved. This can and will be redressed by application of low-temperature stopped-flow spectroscopy in future experiments.

Acknowledgments

This work was supported by Grant 13013-MED of the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich and the Human Frontier Science Program (RGP 0026/2001-M).

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