[11 Redox Role for Tetrahydrobiopterin in Nitric Oxide Synthase Catalysis Low Temperature Optical Absorption Spectral Detection

By Antonius C. F. Gorren, Nicole Bec, Reinhard Lange, and Bernd Mayer

Introduction

Nitric Oxide Synthase

The biosynthesis of nitric oxide (NO) is catalyzed by nitric-oxide synthases (NOS, EC 1.14.13.39; for reviews see Refs. 1-3). These enzymes are homo-dimers, in which each monomer consists of an oxygenase domain and a reductase domain that is homologous to cytochrome P-450 reductase. NOS catalyzes the transformation of l-arginine to l-citrulline and NO in two discrete steps, with /Vc,-hydroxy-l-arginine (NHA) as intermediate. Both reactions take place at a P-450-type heme in the oxygenase domain and consume 1 equivalent of 02. Both reactions also require electrons (two in the first step, and one in the second step), which are supplied by NADPH via two flavin moieties in the reductase domain. Reaction mechanisms proposed for the first step, the conversion of arginine to NHA, are based on the reaction cycle that is thought to describe P-450 catalysis. According to this model, ferric heme is reduced, binds 02, and accepts a second electron. This provides the energy to achieve 0-0 bond scission, expulsion of H20, and formation of oxyferryl heme. This compound hydroxylates arginine bound to the distal heme pocket, resulting in formation of NHA and regeneration of ferric heme. Because of the different electronic requirements of the second step (net consumption of only one NADPH-derived electron), it has generally been assumed that hydroxylation of l-arginine and conversion of NHA to l-citrulline and NO must follow entirely different pathways.

Tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthesis. Although the main functions of the other cofactors and prosthetic groups (heme, FAD, FMN, Ca2+/calmodulin, and Zn2+) are known, the role of BH4 proved to be more contentious, with the argument focusing on whether BH4 is redox active

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2 A. C. F. Gorren and B. Mayer, Biochemistry (Moscow) 63, 870 (1998).

3 S. Pfeiffer, B. Mayer, and B. Hemmens, Angew. Chem. Int. Ed. 38, 1714 (1999).

in NOS catalysis (see Refs. 2-5 for reviews). In the absence of BH4, oxidation of NADPH becomes uncoupled from NO synthesis, and reduction of O2 results in formation of superoxide anion (02~). The well-established structural and allosteric effects of BH4 on NOS do not account for these observations. Moreover, studies with a range of BH4 analogs illustrated the importance of the redox properties of BH4 for NOS catalysis. In the aromatic amino acid hydroxylases, which are the only other enzymes known to use BH4 as a cofactor, BH4 is directly involved in binding and reduction of 02, and it exhibits two-electron redox shuttling between the tetrahydro and quinonoid dihydro species. However, a similar function of BH4 in NOS catalysis can be ruled out, because 5-methyl-BH4, which is unable to undergo reversible two-electron oxidation, is an activator of NO synthesis.6

Cryoenzymology

We decided to use UV-visible (UV-vis) spectroscopy at subzero temperatures to study the reaction of reduced NOS with 02, about which little was known when we started our investigations. Cryoenzymology, the study of enzyme reactions at low temperatures, can provide vital information regarding reaction intermediates that are kinetically or thermodynamically inaccessible by other methods (see Refs. 7-12 for reviews). Preliminary experiments had shown that the reaction of NOS with 02 proceeds too fast for detection of intermediates by standard absorption spectroscopy at ambient temperature. We intended to slow the reaction sufficiently by performing experiments at —30° in a mixed hydro-organic solvent. This technique has been successfully applied to identify intermediates in the reaction of cytochrome P-450 with O2.13-17

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7 T. E. Barman and F. Travers, Methods Biochem. Anal. 31, 1 (1985).

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Experimental Procedures

Equipment

We perform experiments with a Cary 3E (Varian, Palo Alto, CA) spectrophotometer that is adapted for low-temperature studies in a similar way as described in Maurel et al.n These adaptations, which have been carried out in-house, also enable flushing the cuvette holder with dry nitrogen to prevent condensation and formation of ice on cuvette windows and spectrophotometer lenses. For our studies, it has the additional advantage of keeping the cuvette holder anaerobic, and thus of preventing premature oxidation of the samples. The home-built sample compartment contains an aluminum block that serves as a cuvette holder for sample and reference. The temperature of the block is regulated with circulating ethanol that is thermostatted with a Thermo Haake (Karlsruhe, Germany) F3-Q bath with a lower limit of —50°. The temperature of the sample holder is monitored with a thermocouple connected to an AOIP (Evry, France) voltmeter. For thermal insulation the walls of the sample compartment are made of polyvinylchloride, equipped with double quartz windows.

Cryosolvent

An important aspect of cryoenzymology is the choice of a suitable solvent. We have opted for a mixed solvent consisting of aqueous buffer and ethylene glycol in a 1:1 (v/v) ratio. The physicochemical properties of such cryosolvents have been reported.9'19 In the case of phosphate buffer, the pH varies only slightly as a function of temperature. Preliminary studies have shown that this solvent does not affect the spectroscopic properties of NOS at ambient temperature, and the enzyme remains active.20 However, detailed measurements of pterin binding and enzyme activity have revealed that ethylene glycol decreases the affinity of NOS for BH4, such that the dimer appears to bind only one equivalent of BH4.21

Sample Preparation

Samples are prepared in Teflon-capped 3-ml quartz cuvettes at final NOS concentrations of 2-4 ¡lM in total volumes of 1-2 ml, containing 50 mM potassium phosphate (pH 7.4), 1 mM 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 1 mM 2-mercaptoethanol, 0.5 mM EDTA, and 50% ethylene glycol. Each sample is gently flushed with argon for 30 min via two

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19 P. Douzou, G. Hui Bon Hoa, P. Maurel, and F. Travers, in "Handbook of Chemistry and Molecular Biology" (G. D. Fasman, ed.), p. 520. CRC Press, Cleveland, OH, 1976.

20 N. Bee, A. C. F. Gorren, C. Volker, B. Mayer, and R. Lange, J. Biol. Chem. 273, 13502 (1998).

21 A. C. F. Gorren, N. Bee, A. Schrammel, E. R. Werner, R. Lange, and B. Mayer, Biochemistry 39, 11763 (2000).

holes in the Teflon cap. After anaerobiosis, 230 /¿M sodium dithionite (10-20 /il of a freshly prepared anaerobic 23 mM stock solution) is added with a Hamilton syringe and the sample is incubated under an atmosphere of argon for another 30 min. The cuvette is then placed in the cuvette holder at ambient temperature, and an absorbance spectrum is measured to check whether reduction is complete. If so, the temperature is lowered to —30°, which takes about 1 hr. Meanwhile, a 30-ml syringe is filled with 02 and stored at -70° until use. When the temperature of the sample reaches —30°, another spectrum is taken. Subsequently, 2-5 ml of 02 is carefully administered with the precooled syringe. Spectra are measured immediately after 02 addition, and then at certain intervals (usually 2 min) for several hours or until oxidation is complete.

Data Acquisition

Spectra are measured in double-beam mode between 350 and 700 nm, with 1.5-nm slit width. Data acquisition usually is in steps of 0.5 or 1.0 nm with an integration time of 0.5 sec/data point. All spectra are baseline corrected.

Results

The primary goal of our investigations is the identification of reaction intermediate^) in the oxidation of reduced NOS by 02, particularly of the oxy-ferrous complex Fen02, which accumulates under similar conditions in the case of cytochrome P-450. Indeed, we observed formation (within 2 min) of a compound exhibiting a red-shifted absorption maximum at 416/7 nm that we ascribed to oxyferrous (Fen02) heme.20 Most importantly, this intermediate did not accumulate with BH4-containing NOS in the presence of arginine, which implied that under those conditions the reaction cycle continued beyond the Fen02 state. This was unexpected, because the next step in the cycle requires reduction of the oxyferrous complex, and under our reaction conditions no obvious reductant was present. Partly on the basis of this observation we proposed that BH4 can reduce the oxyferrous complex, most likely as a one-electron donor, and that this capacity of enzyme-bound BH4 provides the explanation for the absolute dependence of NO synthesis on the pterin cofactor.20 This hypothesis was confirmed by the fact that under single-turnover conditions NHA was formed in substantial yields only when arginine and BH4 were both present.20

In a subsequent study we made similar observations with NHA instead of arginine.21 With BH4-frce NOS the same 417-nm intermediate was found (Fig. 1), whereas with BH4-containing NOS the reaction ran to completion immediately, resulting in the formation (within 2 min) of an FeIU • NO complex (Fig. 2). This suggests that BH4 has the same function in both reaction cycles. In the presence of the inhibitory redox-inactive pteridine 7,8-BH2 the reaction cycle was halted at the stage of the Fen02 complex with either arginine or NHA, whereas

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