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J. Biol. Chem., Vol. 275, Issue 41, 31581-31587, October 13, 2000

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Nitric-oxide Dioxygenase Activity and Function of Flavohemoglobins

SENSITIVITY TO NITRIC OXIDE AND CARBON MONOXIDE INHIBITION^*

Paul R. Gardner, Anne M. Gardner, and Lori A. Martin

From the Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229

Received for publication, May 15, 2000, and in revised form, July 31, 2000

Yi Dou, Tiansheng Li, and John S. Olson

From the Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005

Received for publication, May 15, 2000, and in revised form, July 31, 2000

Hao Zhu^§, and Austen F. Riggs

From the Section of Neurobiology, School of Biological Sciences, University of Texas, Austin, Texas 78712

Received for publication, May 15, 2000, and in revised form, July 31, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O₂, NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V_max) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme¹ s¹, respectively, at 37 °C with 200 µM O₂. The K_M values for NO are low and range from 0.1 to 0.25 µM. V_max/K_M(NO) ratios of 900-2900 µM¹ s¹ indicate an extremely efficient dioxygenation mechanism. Approximate K_M values for O₂ range from 60 to 90 µM. NO inhibits the dioxygenases at NO:O₂ ratios of 1:100 and makes true K_M(O₂) values difficult to determine. High and roughly equal second order rate constants for O₂ and NO association with the reduced flavoHbs (17-50 µM¹ s¹) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O₂ (K_I(CO) = ~1 µM). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O₂ from inhibitory NO and CO.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Investigations of NO toxicity and defenses in Escherichia coli led to the isolation of an NO-inducible NAD(P)H, O₂, and FAD-dependent NOD¹ activity. This enzyme activity was identified with the flavoHb encoded by the hmp gene (1, 2). More recently, we have shown that the flavoHb efficiently converts NO and O₂ to nitrate via a conventional two electron flavoenzyme mechanism (3). The proposed NOD reaction stoichiometry is described by Equation 1.

(Eq. 1)

A function for flavoHbs in NO dioxygenation and detoxification is supported by a growing body of evidence. FlavoHbs are induced by NO, nitrite, nitrate, and NO-releasing agents in various bacteria (2, 4-9), and these flavoHbs protect bacteria, yeast, and Dictyostelium discoideum against growth inhibition and NO-mediated damage during exposures to authentic NO or NO releasing agents (1, 2, 6, 7, 10-12). Further, O₂ is required for the robust NO scavenging action of flavoHb both in E. coli and in vitro, and O₂ is required for the maximal protection of cells and NO-sensitive aconitases from NO-mediated damage (1, 2). Other flavoHb mechanisms may also protect cells against nitrosothiol or NO toxicity in the absence of O₂. FlavoHbs may reduce NO or denitrosylate toxic nitrosothiols formed from NO, sequester NO or reactive heme, or catalyze other beneficial reactions (6, 10, 13, 14). These additional flavoHb activities and functions require consideration.

We have now investigated the NOD activities of flavoHbs isolated from Saccharomyces cerevisiae, Alcaligenes eutrophus, and E. coli and have compared these activities with other enzymic activities of flavoHbs including NO reductase, NADH oxidase, and FAD reductase activities. Spectra for the various O₂, NO, and CO liganded flavoHbs and the transient kinetics for O₂, NO, and CO binding are reported for flavoHbs and are discussed in relation to the susceptibility of NOD activity to NO and CO inhibition. The kinetics and stoichiometry of the NOD activity indicate a highly specific and efficient dioxygenase mechanism and function for the flavoHbs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- NADPH, FAD, phenylmethylsulfonyl fluoride, and Aspergillus niger glucose oxidase (225 units/mg) were purchased from Sigma. NADH, Aspergillus nitrate reductase, and bovine liver catalase (260,000 units/ml) were from Roche Molecular Biochemicals. Manganese-containing superoxide dismutase (2,700 units/mg) was isolated from E. coli strain DH5 bearing the multi-copy plasmid pD11c (15, 16). Hemin (99.9%) was obtained from Fluka. Ultra-pure O₂ and CO were purchased from Praxair (Bethlehem, PA). NO was obtained from Aldrich.

Purification of FlavoHbs-- S. cerevisiae flavoHb was prepared from strain JM43. Cultures were grown on YPD-galactose medium and treated with antimycin A as described previously (17). Cells from 6 liters of culture (~72 g) were resuspended in 2 volumes of 20 mM potassium phosphate, pH 7.6, containing 1 µM phenylmethylsulfonyl fluoride, and cells were lysed in a French press. Lysates were clarified by centrifugation at 16,000 × g, and cell-free extracts were applied to a 2.5 × 10-cm DEAE-Sepharose CL-6B column (Sigma). After washing with 5 column volumes of 20 mM potassium phosphate, pH 7.6, and 50 mM KCl, flavoHb was eluted with a linear gradient of 50-150 mM KCl. Fractions with a 405 nm:280 nm ratio larger than 0.06 were pooled and concentrated on a YM10 membrane (Amicon). FlavoHb was then separated by gel electrophoresis in 10% polyacrylamide containing 25 mM Tris/192 mM glycine, pH 8.8, at 4 °C. The yellowish flavoHb band was excised and eluted with Electro-Eluter Model 422 (Bio-Rad). A. eutrophus flavoHb was purified from cell pellets provided by Dr. Bärbel Friedrich (University of Berlin, Germany) (14) as described above. E. coli flavoHb was purified from anaerobic nitrate-induced cultures of RB9060 containing the multi-copy plasmid pAlter + hmp (3). E. coli flavoHb was reconstituted with hemin as described (3). FlavoHb purities were judged to be greater than 95% by reverse phase HPLC and SDS-polyacrylamide gel electrophoresis analysis.

FlavoHb Activity Assays-- NOD activity was measured using a NO electrode (World Precision Inst.) at 37 °C in 2 ml of 50 mM potassium phosphate, pH 7.8, containing 100 µM EDTA, 1 µM NO, 1 µM FAD, 200 µM O₂, and 100 µM NADH unless specified otherwise (3). Saturated NO (2 mM) was prepared as described previously (18). Saturated CO (1 mM) was prepared by vigorously stirring water in a septum-sealed vial under a stream of 99.99% CO (Praxair) at room temperature (19). Reaction mixtures were first scrubbed with 99.999% N₂ when the effects of O₂ concentration were measured. Saturated O₂ solution (1.14 mM) was prepared by vigorously stirring reaction buffer in a rubber septum-sealed vial under 99.99% O₂ (Praxair) at room temperature. The effect of FAD and CO on NOD activity was measured following repetitive additions of 2 nmol of NO and the stepwise removal of NO by 0.12-1.2 pmol of flavoHb in a 2-ml reaction at 37 °C. NOD activities were calculated with a correction for background rates of NO decomposition for each assay condition. NADH oxidase activity was followed at 340 nm in a 0.5-ml reaction mix containing 50 mM potassium phosphate, pH 7.8, 100 µM EDTA, 100 µM NADH, and 1 µM FAD at 37 °C. NO reductase activity was measured using the NO electrode in an anaerobic 2-ml reaction at 37 °C containing 50 mM potassium phosphate, pH 7.8, 0.1 mM EDTA, 1 µM FAD, 10 mM glucose, 8 units of glucose oxidase, 260 units of catalase, 100 µM NADH, and 5 µM NO. NO and flavoHb were added following 6 min of O₂ depletion, and NO decomposition rates were determined for 1 µM NO. FAD reductase activity was assayed at 450 nm in a 1-ml reaction mixture prepared as described for the measurement of NO reductase activity except that NO was omitted and 20 µM FAD was included.

Cofactor and Protein Assays-- Heme was determined using the alkaline pyridine method (20). FAD was released from flavoHb by boiling for 3 min and was determined from the fluorescence at 520 nm with excitation at 460 nm (21). Alternatively, heme and FAD were adsorbed on the reverse-phase C18 HPLC column (SynChrom) in 0.1% trifluoroacetic acid in H₂O and were separated with a gradient of acetonitrile in 0.1% trifluoroacetic acid. Heme and FAD were eluted with 35-40% and 10-15% acetonitrile, respectively. FAD and heme standards were employed for the analysis. E = 6,220 M¹ cm¹ at 340 nm was used for the calculation of NAD(P)H concentration. The percentage of reduction of NAD(P)H preparations was determined from the absorbance ratio for 340 nm and 260 nm using 14,250 M¹ cm¹ at 260 nm (22). NADH and NADPH were 95% reduced. Protein was measured using the dye binding assay with bovine serum albumin as the standard (23).

Measurements of Rate Constants for Ligand Binding-- All measurements were made at 20 °C in 100 mM potassium phosphate, pH 7.0, and 0.3 mM EDTA for comparison with previous Mb and Hb studies. Recombinant sperm whale Mb was included as a control. FlavoHb and Mb ligand complexes were prepared, and stopped flow and laser photolysis measurements of ligand binding were made as described previously (3, 19).

Nitrate, Nitrite, O₂, and NADH Measurements-- Nitrate and nitrite were measured using the Griess reagent and nitrate reductase (24). O₂ consumption was measured with a Clark-type O₂ electrode using a value of 200 µM O₂ for air-saturated buffer at normal atmospheric pressure and 37 °C (25). NADH oxidation was monitored at 340 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Spectra of FlavoHbs-- Visible light absorbance by FAD and heme confers an orangish brown color on flavoHbs that is altered by reduction and ligand binding. The heme spectra and absorbance maxima are grossly similar for the ligand complexes and redox forms of various flavoHbs (3, 13, 21, 26-29). We measured absorbance spectra of purified S. cerevisiae, A. eutrophus, and E. coli flavoHbs under identical conditions to evaluate the degree of spectral identity. With O₂ binding, the NADH-reduced flavoHbs display heme spectra that are typical of oxyHbs (Fig. 1). The flavoHbO₂ complexes show similar wavelength maxima for the Soret and , bands (Table I). Notable differences are observed in the spectra attributable to the oxidized flavin (470 nm) and to the free NADH (340 nm). The E. coli flavoHb/NADH mixture shows greater absorbance at 470 nm and less absorbance at 340 nm; moreover, spectra must be recorded rapidly to observe the E. coli flavoHbO₂ complex because of the NADH oxidase activity (3, 28-30). The other flavoHbO₂ complexes do not have as high an oxidase activity (see Table V) but still deplete O₂ and NADH in minutes at the 1-10 µM flavoHb concentrations used. A. eutrophus flavoHbO₂ shows a relatively low absorbance at 625 nm. Spectra for flavoHbNO and CO complexes and the reduced and oxidized forms reveal similar heme absorbance peaks (Table I). These spectra demonstrate the formation of both the flavoHbO₂ and flavoHbNO complexes that are required for flavoHb-catalyzed NO dioxygenation (2, 3, 31, 32) and NO reduction (13), respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Absorbance spectra of flavoHb(Fe2+)-O2 complexes. Reduced S. cerevisiae (line 1), A. eutrophus (line 2), and E. coli (line 3) flavoHb(Fe2+)-O2 complexes (10 µM heme) were prepared with NADH and O2 and were scanned as described under "Materials and Methods." The E. coli flavoHb was reconstituted with bovine heme and contained 0.95 mole fraction of heme. S. cerevisiae and A. eutrophus flavoHbs contained 0.95 mole fraction of heme as isolated. The mole fractions of FAD for the E. coli, S. cerevisiae, and A. eutrophus flavoHbs were 0.41, 0.44, and 0.44, respectively.

O₂, NO, and CO Binding Kinetics of FlavoHbs-- O₂ and NO binding kinetics play a critical role in regulating the catalytic rate of the E. coli NOD activity (3). Thus, we examined rates of ligand binding to the S. cerevisiae and A. eutrophus proteins and compared these rates with those for E. coli flavoHb and recombinant sperm whale Mb. The NAD(P)H-reduced flavoHbs show rate constants for O₂ association in the range of 17-50 µM¹ s¹, which are only slightly greater than those reported for mammalian Mbs (Table II). The O₂ dissociation rate constants for all three flavoHbs are very small, 0.2-0.6 s¹, compared with that for MbO₂, 15 s¹. The net result is that all three flavoHbs show O₂ association equilibrium constants that are 20-200 times greater than that for Mb. The association rate constants for NO binding to reduced flavoHb(Fe²⁺) and Mb(Fe²⁺) vary by <3-fold and are in the same range as the rate constants for O₂ binding, 10-30 µM¹ s¹. Greater differences are seen in the ligand binding rate constants for NO binding to the oxidized flavoHb(Fe³⁺)s and for CO binding to the reduced proteins. All three flavoHb(Fe³⁺)s have unusually high NO association rate constants compared with that for Mb(Fe³⁺). The NO dissociation rate constants are also higher for flavoHb(Fe³⁺) than Mb(Fe³⁺). This result suggests that water is not bound or is only weakly coordinated to the Fe³⁺ atom in the oxidized forms. This conclusion is supported by the positions of the Soret peaks of the Fe³⁺(met) forms that are all significantly blue shifted compared with those for human metHb and metMb.

The E. coli flavoHb shows a much greater rate of CO binding than those observed for A. eutrophus and S. cerevisiae enzymes (Table II and Fig. 2). The majority of the absorbance change observed after flash photolysis of E. coli flavoHbCO occurs rapidly with an apparent bimolecular rate constant of ~20 µM¹ s¹, whereas absorbance changes for CO rebinding to A. eutrophus and S. cerevisiae flavoHbs show a much smaller k'_CO value equal to 0.1-0.5 µM¹ s¹. As shown in Fig. 2, a slow phase is observed for CO binding to the E. coli enzyme and the k'_CO value for this phase is similar to that of the other flavoHbs, ~0.5 µM¹ s¹. In contrast to CO binding, all three flavoHbs have high O₂ association rate constants, low O₂ dissociation rate constants, and consequently high O₂ affinities for the flavoHb(Fe²⁺), properties shown to be important for the efficient NOD activity of the E. coli flavoHb (3). However, the differences in NO and CO binding suggest that the heme pockets of the flavoHbs differ.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   CO rebinding to flavoHbs. NADH-reduced A. eutrophus (line 1) and E. coli (line 2) flavoHbs were equilibrated with 1 atm CO in 0.1 M phosphate, pH 7.0, at 20 °C and were photolyzed with a 300-ns excitation pulse.

Steady-state Kinetic Constants for the NOD Activities of FlavoHbs-- Extensive amino acid sequence identities and spectral and ligand binding similarities among the flavoHbs strongly suggested a similar capacity for NO dioxygenation. The S. cerevisiae flavoHb was also recently shown to consume NO in an O₂-dependent reaction in whole cells, thus supporting a common dioxygenase and NO detoxification function (11). Nevertheless, it remained to be determined whether various flavoHbs share a similar NOD activity. The flavoHbs show NOD activities with similar dependences upon O₂ concentration as expected for similar O₂ ligand binding constants (Table II). At 0.75 µM NO, 37 °C and with 200 µM NADH as reducing substrate, the S. cerevisiae, A. eutrophus, and E. coli flavoHbs show apparent K_M values for O₂ of 60, 80, and 90 µM, respectively (Fig. 3, A-C, lines 1). Further, each flavoHb is competitively inhibited by CO as expected for a competition between O₂ and CO for binding the reduced heme iron atom (Table II). The K_I values for CO for the S. cerevisiae, A. eutrophus, and E. coli flavoHbs are approximately 1, 0.5, and 1 µM, respectively (Fig. 3). Inhibition by CO is rapid and reversible as demonstrated by a constant rather than progressive inhibition of NOD activity during repetitive catalytic turnover (Fig. 4). The data further support a mechanism of NO dioxygenation involving the reaction of NO with a flavoHbO₂ complex (3) and suggest greater similarities of CO affinities of flavoHbs during catalytic turnover than those measured by rapid kinetics (Table II).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   O2 dependences and CO sensitivities of the NOD activities of flavoHbs. NOD activity of S. cerevisiae (A), A. eutrophus (B), and E. coli (C) flavoHbs was measured at 37 °C with various O2 concentrations in the presence of 0 µM CO (line 1), 0.5 µM CO (line 2), 1 µM CO (line 3), or 2 µM CO (line 4) in reactions containing 0.75 µM NO, 200 µM NADH, and 1 µM FAD.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   CO inhibition of NOD activities during turnover. NOD activity of E. coli flavoHb was measured with 0 µM CO (line 1), 0.5 µM CO (line 2), 1 µM CO (line 3), 2 µM CO (line 4), and 4 µM CO (line 5) following repetitive additions of 1 µM NO as described under "Materials and Methods." One catalytic cycle represents the two-electron reaction measured by the decomposition of two NO molecules and was calculated relative to the heme content of flavoHb.

Each of the flavoHbs shows a normal saturation with NO at 200 µM O₂ (Fig. 5, A-C, lines 1). The apparent K_M values for NO for the S. cerevisiae, A. eutrophus, and E. coli NOD activities are 0.13, 0.10, and 0.25 µM, respectively (Table III). However, at 25 and 50 µM O₂, inhibition by NO is apparent at 0.25 and 0.5 µM NO concentrations, respectively (Fig. 5, A-C, lines 2 and 3), and inhibition by NO appears more pronounced for the S. cerevisiae and A. eutrophus flavoHbs. The ability of NO to inhibit NOD activities at NO:O₂ ratios greater than 1:100 should elevate the values of K_M(O₂) determined from the data in Fig. 3. However, analyses of the O₂ dependence of the E. coli NOD activity at low NO concentrations have indicated that the effect of NO inhibition on K_M(O₂) determination under these conditions is small (3).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   NO dependences of the NOD activities of flavoHbs. NOD activity of S. cerevisiae (A), A. eutrophus (B), or E. coli (C) flavoHb was measured at 37 °C with 100 µM NADH, 1 µM FAD, and 200 µM O2 (line 1), 50 µM O2 (line 2), or 25 µM O2 (line 3) with varying concentrations of NO as described under "Materials and Methods."

The flavoHbs show large differences in their dependence upon NADH and NADPH for NOD catalysis (Table III). The maximal turnover rates (V_max) calculated from plots of 1/v versus 1/[NAD(P)H] with saturating NADH or NADPH are also summarized in Table III. It is noteworthy that the A. eutrophus flavoHb does not utilize NADPH as a reducing substrate and that the S. cerevisiae and E. coli flavoHbs utilize either NADH or NADPH. The results are consistent with earlier reports of the specificity of various A. eutrophus flavoHb activities for NADH (27). Importantly, none of the NOD activities was significantly affected by superoxide dismutase (1 mg/ml) under standard assay conditions indicating a limited role for free O₂ in flavoHb-catalyzed decomposition of NO.

We also examined the effect of temperature on the NOD activities. The E. coli NOD activity decreases ~3-fold in a shift from 37 to 20 °C, whereas the S. cerevisiae and A. eutrophus activities decrease by 21- and 16-fold, respectively (Table IV). These results indicate large effects of temperature on NOD catalysis and suggest possible differences in the rate-limiting flavin to heme electron transfer step between the flavoHbs (3). Differences in temperature effects may also be explained in part by relative differences in NO or O₂ saturation.

FAD Dependence of the NOD Activities of FlavoHbs-- The A. eutrophus flavoHb structure shows one FAD and one heme per protein molecule (33); however, some preparations of flavoHb are deficient in one or both cofactors following isolation (2, 3, 21). Our preparations of S. cerevisiae, A. eutrophus, and E. coli flavoHbs contained 28-44% of the predicted FAD. A 2-min preincubation with FAD increases the initial NOD activity for each flavoHb by ~7-30% (Fig. 6). The effect is largest for E. coli flavoHb, which is the most FAD-deficient of the three (0.28 mole fraction). In the absence of added FAD, each of the flavoHbs loses 50% of its NOD activity within ~5,000 two-electron catalytic cycles (Fig. 6, lines 4-6). For E. coli flavoHb, this activity loss is even faster with lower but still saturating, concentrations of NADH or with NADPH (1-3). In the presence of 1 µM FAD, the E. coli and S. cerevisiae NOD activities are more stable (lines 1 and 3). In contrast, the A. eutrophus enzyme slowly loses activity even with added FAD (line 2).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effects of FAD on the NOD activities of flavoHbs. NOD activity of 1.2 pmol of S. cerevisiae flavoHb (lines 1 and 4), 0.48 pmol of A. eutrophus flavoHb (lines 2 and 5), 0.79 pmol of E. coli flavoHb (lines 3 and 6) was measured at 37 °C with (lines 1-3) or without (lines 4-6) 1 µM FAD as described under "Materials and Methods." Reactions were initiated with 1 µM NO, following a brief 2-min preincubation of flavoHbs with or without FAD plus 100 µM NADH and 200 µM O2. S. cerevisiae, A. eutrophus, and E. coli flavoHbs contained 0.44, 0.44, and 0.28 mole fractions of FAD and ~1.0, ~1.0, and 0.42 mole fractions of heme, respectively. Cycles were calculated relative to flavoHb heme content.

The results demonstrate the requirement of a free pool of FAD for sustained and maximal NOD activity of flavoHbs during the course of a typical NOD activity assay and suggest dissociation and reassociation of bound FAD during enzyme turnover. These results, together with the FAD deficiency of the flavoHb preparations, warranted the inclusion of FAD in all flavoHb activity assays. In addition, the data indicate that NO inhibition of the NOD activity is effectively reversible, at least for the E. coli and S. cerevisiae flavoHbs, because there is no significant loss of activity following repetitive NO additions (1 µM) and a total of >10,000 two electron turnover cycles (Fig. 6, lines 1 and 3).

Other Enzymic Activities of FlavoHbs-- We investigated three other enzymic activities of the flavoHbs and compared their turnover rates with those for NO dioxygenation. Each flavoHb shows the anaerobic NO reductase activity reported for E. coli flavoHb (6, 13) (Table V). The E. coli flavoHb NO reductase activity is ~4-fold higher than that of the other flavoHbs but is still several orders of magnitude lower than that of the NOD activity. Each flavoHb also displays the NADH oxidase activity reported for E. coli flavoHb (28-30). The E. coli NADH oxidase activity is ~7-fold higher than that of the other flavoHbs. This higher NADH oxidase activity accounts for the rapid depletion of NADH by E. coli flavoHb observed in spectra of flavoHbO₂ (Fig. 1). The data also reveal comparable anaerobic FAD reductase activities for the flavoHbs (34).

Stoichiometry of the FlavoHb Reaction-- The stoichiometry of the flavoHb-catalyzed decomposition of NO was measured using the reaction conditions now established for optimal NOD activity. FlavoHb converted 40 nmol of NO, 39.1 nmol of O₂, and 19.9 nmol of NADH to 39.6 nmol of nitrate. The 2-ml reaction contained an initial 100 µM NADH, 200 µM O₂, and 1 µM FAD in 50 mM potassium phosphate buffer, pH 7.8, and 0.1 mM EDTA at 37 °C with 8 pmol of E. coli flavoHb and was primed with 10 sequential additions of 2 µM NO. Nitrite was not a significant product of the reaction.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Various functions for Hbs, Mbs, and the two-domain flavoHbs have been postulated based upon their intrinsic physical properties (35-38). By definition, members of this Hb superfamily are globular, contain heme, and bind O₂ reversibly (39). Partial transfer of the ferrous heme electron to O₂ strengthens the Hb(Fe²⁺)O₂ bond and may especially suit Hbs for O₂ transport, storage and sequestration functions (40). The Hb(Fe²⁺)O₂ complex may also be optimized to catalyze the oxidation or oxygenation of suitable substrates (36). Indeed, many oxyHbs catalyze a variety of (per)oxidations (38, 41-44) and rapidly dioxygenate NO (31, 32). A (per)oxidase or peroxygenase function for Hbs is attractive because a dehalo-peroxygenase/peroxidase activity isolated from a marine polychaete annelid has been found to share 20.6% amino acid sequence identity with sea hare Mb (45). Recent proposals of specialized functions have also included a reductive removal of toxic O₂ by the perienteric Ascaris suum Hb (46).

A function for flavoHb and Hbs with associated reductases was suggested with the identification of the efficient NOD activity in E. coli with flavoHb (1-3). We have now demonstrated similar NOD activities of the A. eutrophus and S. cerevisiae flavoHbs. The results extend the range of the NOD function of the ancient flavoHbs from bacteria to yeast (17). These NOD activities have high turnover rates and low K_M values for NO (Table III). V_max/K_M(NO) ratios for NOD activities at 37 °C and a normoxic O₂ level of 200 µM are 900-2,900 µM¹ s¹. These apparent bimolecular rate constants for flavoHb-catalyzed NO dioxygenation are only 2-6-fold lower than the diffusion-limited second order rate constant for the reaction of NO with free superoxide (47, 48) and are more than 20-fold higher than those reported for the mammalian oxyMb and oxyHb (32). Further, maximal NOD turnover rates exceed the turnover rates measured for NO reduction, NADH oxidation, and FAD reduction by several orders of magnitude (Table V). The reaction stoichiometry supports the proposed NO dioxygenase mechanism described by Equation 1 (2, 3).

Previous approximations of the flavoHb reaction stoichiometry have also supported a dioxygenase mechanism for NO metabolism (6). However, the high concentrations of NO (100 µM) and flavoHb (~0.5 µM) used in these early studies are likely to have provided conditions for NO inhibition, NO reduction, reactions leading to the formation of nitrite, and nonstoichiometric nitrate, NADH and O₂ ratios. NO dioxygenation thus appears to be a highly specific and well adapted enzymic function for the flavoHbs. FlavoHbs may significantly control NO toxicity and signaling functions in a variety of organisms.

Competition between NO and O₂ for binding to reduced flavoHb (Table II) inhibits NOD activities at NO:O₂ ratios 1:100 (Fig. 5). CO shows inhibitory effects comparable with those of NO with 50% inhibition occurring at CO:O₂ ratios of 1:60. Given the sensitivity of the NOD reaction to NO and CO inhibition, NO and CO may inhibit NOD activity in vivo. NOD activity inhibition may affect the survival of organisms exposed to toxic NO and CO produced by the cells of many tissues of higher organisms (49-51). Furthermore, because NO and CO are abundantly produced by organic combustion and decay reactions, NO and CO inhibition of the NOD activity of the ancient flavoHbs may have been an important factor in the evolution of the ligand discrimination properties of Hbs.

The structural causes of the differences in CO binding between the flavoHbs and the heterogeneity seen in the E. coli flavoHb traces are not clear. There are two possibilities. The proteins may exist in alternative conformations, one rapidly reacting and the other slowly reacting. Based on the low K_I for CO seen in the NOD steady-state assays, the more rapidly reacting conformer appears to occur during catalysis. The cause of the rate differences could be explained by differing extents of hydration in the distal pockets or changes in iron reactivity, but no structural data are available to allow discrimination between these possibilities. Alternatively, the differences may be related to the extent of reduction of the CO complexes. If only 1 or 2 electrons are present, they may redistribute to the flavin after photolysis, causing both the fraction of reduced iron and, hence, reactivity toward CO, to decrease markedly. A similar mechanism could explain the high K_M values for O₂ observed in the steady-state assays, where the enzyme cycles between the two, one, and oxidized enzyme species (3). The competition between the flavin and iron for electrons could cause a significantly lower reactivity toward O₂ as well. In our transient kinetic measurements with the reduced flavoHbO₂ complex, the enzymes are almost certainly in the three-electron reduced state. The initial reduced state will contain two electrons from NADH. When autooxidation creates the one electron reduced species, flavoHb is immediately reduced by the excess of NADH present. In the case of CO complexes, NADH reduction in the absence of O₂ can only create the two-electron reduced species.

The reasons for the FAD deficiency of flavoHb preparations and the apparent loss of bound FAD during catalytic turnover remain to be elucidated. Nevertheless, these and other data suggest that FAD readily dissociates from the flavoHbs. Substoichiometric increases in the NOD activities of the FAD-deficient flavoHbs with added FAD (Fig. 6) may be explained by an incomplete complement of heme or the introduction of trace amounts of FAD with NADH that has been prepared from biological sources. The low stimulation of activity may also reflect nonoptimal conditions for FAD association.

FlavoHb and Hb genes and proteins are found in diverse microorganisms (5, 8-10, 12, 17, 52-59) including D. discoideum (AB025583 and AB025584), Pyricularia grisea (AA415144), Erwinia chrysanthami (O47266), Xylella fastidiosa (AE003859.1), Deinococcus radiodurans (AE001863), Salmonella typhimurium (AF020388.1), Bacillus subtilis (P49852), Bacillus halodurans (AB024563), Campylobacter jejuni (AL139079), Schizosaccharomyces pombe (AL132779.2), Vibrio parahaemolyticus (P40609), Fusarium oxysporum (AB016807), Pichia norvegensis (Q03331), Botrytis cinerea (AL116429.1 and AL115888.1), Vitreoscilla stercoraria (P04252), Aquifex aeolicus (AE000678), and Mycobacterium bovis (AF130980 and AF213450). Hbs with poorly defined functions are also expressed in various plant and animal tissues (35, 60). Trademarks of flavoHbs, including amino acid sequences, spectra, and ligand binding kinetics, may point to a NOD function for this type of heme protein. Highly conserved and unique flavoHb and Hb heme pocket residues Tyr(B10) and Gln(E7) may especially signify a NOD function. The large O₂ association rate constants and small O₂ dissociation rate constants of flavoHb, conferred by the Tyr(B10) hydroxyl, provide the relatively stable Fe²⁺-O₂ intermediate required for the enzymic NO dioxygenation (3).

NO reductase, nitrosothiol denitrosolase, NADH oxidase, FAD reductase, and other proposed activities of the flavoHbs (6, 13) also require consideration in the evaluation of flavoHb function. Thus, although an O₂ requirement for protection against NO damage has been demonstrated for flavoHb in E. coli (1, 2), flavoHbs have provided comparable growth protection against NO donor compound and nitrosothiol toxicity in anoxic S. typhimurium and S. cerevisiae cultures (10, 11). These anaerobic protective effects of flavoHb have suggested the participation of additional flavoHb activities, functions, and mechanisms. An anaerobic NO reduction function has been suggested (6, 10, 11, 13, 14). However, the prevalence of separate and efficient NO-inducible anaerobic NO reductases in E. coli and other organisms including A. eutrophus (1, 61) coupled with the extremely low NO reductase activities of flavoHbs (Table V) suggests that the anaerobic protective effects of flavoHbs are not related to the reduction of NO to the nitroxyl anion. Moreover, given the nanomolar O₂ dissociation equilibrium constants of flavoHbs (Table II) and the high NOD turnover rates, it is clear that trace O₂ contamination may produce a significant NOD activity in growth protection experiments. In addition, greater knowledge of the differences between authentic NO, nitrosothiol, acidified nitrite, and NO donor compound toxicities and the physiological significances of these toxicities is required for a full understanding of the NO detoxification function of flavoHbs.

The finding that NOD activity is conserved among the 1.8 billion-year-old flavoHbs (17) suggests that the ancestral Hb originated to help protect cells from the damaging effects of ambient NO and O₂ by combining them to form nontoxic nitrate. A nitrate production function may have also been advantageous during the ~1.5 billion-year period between the origin of oxygen-releasing photosynthesis and the advent of energy-yielding aerobic respiration. Nitrate would have yielded energy by serving as an anaerobic terminal electron acceptor at the same time that toxic NO and O₂ were removed. More stable O₂-transporting Hbs and O₂-storing Mbs would have only evolved much later when the atmosphere became rich in O₂ and large multicellular animals became possible because of the energy O₂ provided via an efficient O₂ supply system and respiration (62). The earliest Hbs may have served primarily as detoxifying agents along with superoxide dismutases, catalases, peroxidases, and cytochrome P-450s (60).

	ACKNOWLEDGEMENTS

We thank Dr. Bärbel Friedrich and her laboratory for generously supplying A. eutrophus cell pellets. We thank Prof. Helmut Beinert and reviewers of an earlier draft of the manuscript for comments. We thank Dr. Peter Rich for carrying out some early experiments.

	FOOTNOTES

^* This work was supported in part by American Heart Association Grant 9730193N (to P. R. G.), a Children's Hospital Research Foundation trustee grant (to P. R. G.), United States Public Health Service Grants GM35649 (to J. S. O.), HL47020 (to J. S. O.), and GM35847 (to A. F. R.), National Science Foundation Grant MCB9511759 (to A. F. R.), and Grant C-612 (to J. S. O.) from the Robert A. Welch Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 513-636-4885; Fax: 513-636-4892; E-mail: gardp0@chmcc.org.

^§ Portions of this paper are based on a Ph.D. dissertation at the University of Texas in 1994. Present address: Hematology Div., Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115.

Published, JBC Papers in Press, August 1, 2000, DOI 10.1074/jbc.M004141200

	ABBREVIATIONS

The abbreviations used are: NOD, nitric-oxide dioxygenase; flavoHb, flavohemoglobin; Hb, hemoglobin; Mb, myoglobin; HPLC, high pressure liquid chromatography.

	REFERENCES

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