Assessing the impact of denitrifier-produced nitric oxide on other bacteria

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low muM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.     

Catalysis of nitrosyl transfer by denitrifying bacteria is facilitated by nitric oxide

Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO. Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO.     

A conserved tryptophan in nitric oxide synthase regulates heme-dioxy reduction by tetrahydrobiopterin.

In nitric oxide synthase (NOS), (6R)-tetrahydrobiopterin (H(4)B) binds near the heme and can reduce a heme-dioxygen intermediate (Fe(II)O(2)) during Arg hydroxylation [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. A conserved Trp engages in aromatic stacking with H(4)B, and its mutation inhibits NO synthesis. To examine how this W457 impacts H(4)B redox function, we performed single turnover reactions with the mouse inducible NOS oxygenase domain (iNOSoxy) mutants W457F and W457A. Ferrous mutants containing Arg and H(4)B were mixed with O(2)-containing buffer, and then heme spectral transitions, H(4)B radical formation, and Arg hydroxylation were followed versus time. A heme Fe(II)O(2) intermediate was observed in W457A and W457F and had normal spectral characteristics. However, its disappearance rate (6.5 s(-1) in W457F and 3.0 s(-1) in W457A) was slower than in wild-type (12.5 s(-1)). Rates of H(4)B radical formation (7.1 s(-1) in W457F and 2.7 s(-1) in W457A) matched their rates of Fe(II)O(2) disappearance, but were slower than radical formation in wild-type (13 s(-1)). The extent of H(4)B radical formation in the mutants was similar to wild-type, but their radical decayed 2-4 times faster. These kinetic changes correlated with slower and less extensive Arg hydroxylation by the mutants (wild-type > W457F > W457A). We conclude that W457 ensures a correct tempo of electron transfer from H(4)B to heme Fe(II)O(2), possibly by stabilizing the H(4)B radical. Proper control of these parameters may help maximize Arg hydroxylation and minimize uncoupled O(2) activation at the heme.     

Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction.

Five Tn5 mutants of Pseudomonas fluorescens AK-15 deficient in dissimilatory reduction of nitrite were isolated and characterized. Two insertions occurred inside the nitrite reductase structural gene (nirS) and resulted in no detectable nitrite reductase protein on a Western immunoblot. One mutant had Tn5 inserted inside nirC, the third gene in the same operon, and produced a defective nitrite reductase protein. Two other mutants had insertions outside of this nir operon and also produced defective proteins. All of the Nir- mutants characterized showed not only loss of nitrite reductase activity but also a significant decrease in nitric oxide reductase activity. When cells were incubated with 15NO in H2(18)O, about 25% of the oxygen found in nitrous oxide exchanged with H2O. The extent of exchange remained constant throughout the reaction, indicating the incorporation of 18O from H2(18)O reached equilibrium rapidly. In all nitrite reduction-deficient mutants, less than 4% of the 18O exchange was found, suggesting that the hydration and dehydration step was altered. These results indicate that the factors involved in dissimilatory reduction of nitrite influenced the subsequent NO reduction in this organism.     

Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide.

Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen. Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2. Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance. Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation. NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification). The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate. A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis. Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1.     

Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen

The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N(2)O (2NO + 2H(+) + 2e(-) --> N(2)O + H(2)O), which is an obligatory step in the sequential reduction of nitrate to dinitrogen known as denitrification. NOR has four redox-active cofactors, namely, two low-spin hemes c and b, one high-spin heme b(3), and a non-heme iron Fe(B), and belongs to same superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electron acceptor; this catalytic activity was investigated in this study. We show that the product in the steady-state reduction of oxygen is water. A single turnover of the fully reduced NOR with oxygen was initiated using the flow-flash technique, and the progress of the reaction monitored by time-resolved optical absorption spectroscopy. Two major phases with time constants of 40 micros and 25 ms (pH 7.5, 1 mM O(2)) were observed. The rate constant for the faster process was dependent on the O(2) concentration and is assigned to O(2) binding to heme b(3) at a bimolecular rate constant of 2 x 10(7) M(-)(1) s(-)(1). The second phase (tau = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protons from the bulk solution. The rate constant for this phase shows a pH dependence consistent with rate limitation by proton transfer from an internal group with a pK(a) = 6.6. This group is presumably an amino acid residue that is crucial for proton transfer to the catalytic site also during NO reduction.     

Nitric oxide-dependent proton translocation in various denitrifiers.

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.     

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.     

Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens.

The influence of low redox potentials and H2S on NO and N2O reduction by resting cells of denitrifying Pseudomonas fluorescens was studied. Hydrogen sulfide and Ti(III) were added to achieve redox potentials near -200 mV. The control without reductant had a redox potential near +200 mV. Production of 13NO, [13N]N2O, and [13N]N2 from 13NO3- and 13NO2- was followed. Total gas production was similar for all three treatments. The accumulation of 13NO was most significant in the presence of sulfide. A parallel control with autoclaved cells indicated that the 13NO production was largely biological. The sulfide inhibition was more dramatic at the level of N2O reduction; [13N]N2O became the major product instead of [13N]N2, the dominant product when either no reductant or Ti(III) was present. The results indicate that the specific action of sulfide rather than the low redox potential caused a partial inhibition of NO reduction and a strong inhibition of N2O reduction in denitrifying cells.     

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.     

Influence of inhaled nitric oxide on gas exchange during normoxic and hypoxic exercise in highly trained cyclists.

This study tested the effects of inhaled nitric oxide [NO; 20 parts per million (ppm)] during normoxic and hypoxic (fraction of inspired O(2) = 14%) exercise on gas exchange in athletes with exercise-induced hypoxemia. Trained male cyclists (n = 7) performed two cycle tests to exhaustion to determine maximal O(2) consumption (VO(2 max)) and arterial oxyhemoglobin saturation (Sa(O(2)), Ohmeda Biox ear oximeter) under normoxic (VO(2 max) = 4.88 +/- 0.43 l/min and Sa(O(2)) = 90.2 +/- 0.9, means +/- SD) and hypoxic (VO(2 max) = 4.24 +/- 0.49 l/min and Sa(O(2)) = 75.5 +/- 4.5) conditions. On a third occasion, subjects performed four 5-min cycle tests, each separated by 1 h at their respective VO(2 max), under randomly assigned conditions: normoxia (N), normoxia + NO (N/NO), hypoxia (H), and hypoxia + NO (H/NO). Gas exchange, heart rate, and metabolic parameters were determined during each condition. Arterial blood was drawn at rest and at each minute of the 5-min test. Arterial PO(2) (Pa(O(2))), arterial PCO(2), and Sa(O(2)) were determined, and the alveolar-arterial difference for PO(2) (A-aDO(2)) was calculated. Measurements of Pa(O(2)) and Sa(O(2)) were significantly lower and A-aDO(2) was widened during exercise compared with rest for all conditions (P < 0.05). No significant differences were detected between N and N/NO or between H and H/NO for Pa(O(2)), Sa(O(2)) and A-aDO(2) (P > 0.05). We conclude that inhalation of 20 ppm NO during normoxic and hypoxic exercise has no effect on gas exchange in highly trained cyclists.     

Control of nitric oxide synthase dimer assembly by a heme-NO-dependent mechanism

Homodimer formation is a key step that follows heme incorporation during assembly of an active inducible nitric oxide synthase (iNOS). In cells, heme incorporation into iNOS becomes limited due to interaction between self-generated NO and cellular heme [Albakri, Q., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 5414-5421]. Here we investigated if NO can regulate at points downstream in the process by inhibiting dimerization of heme-containing iNOS monomer. Heme-containing monomers were generated by treating iNOS dimer or iNOS oxygenase domain dimer (iNOSoxy) with urea. Both monomers dimerized when incubated with Arg and 6R-tetrahydrobiopterin (H4B), as shown previously [Abu-Soud, H. M., Loftus, M., and Stuehr, D. J. (1995) Biochemistry 34, 11167-11175]. The NO-releasing drug S-nitrosyl-N-acetyl-D,L-penicillamine (SNAP; 0-0.5 mM) inhibited dimerization of iNOS monomer in a dose- and time-dependent manner, without causing heme release. SNAP-pretreated monomer also did not dimerize in response to H4B plus Arg. SNAP converted Arg- and H4B-free iNOS dimer into monomer that could not redimerize, but had no effect on iNOS dimer preincubated with Arg and H4B. Anaerobic spectral analysis showed that NO from SNAP bound to the ferric heme of iNOSoxy monomer or dimer. Adding imidazole as an alternative heme ligand prevented SNAP from inhibiting iNOS monomer dimerization. We conclude that NO and related species can block iNOS dimerization at points downstream from heme incorporation. The damage to heme-containing monomer results from a reaction with the protein and appears irreversible. Although dimeric structure alone does not protect, it does enable Arg and H4B to bind and protect. Inhibition appears mediated by NO coordinating to the ferric heme iron of the monomer.     

Isotope labeling studies on the mechanism of N-N bond formation in denitrification

The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2. The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme. Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite. Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions. The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained. The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8-11).     

The generation of free radicals by nitric oxide synthase

Nitric oxide synthase (NOS) is an example of a family of heme-containing monooxygenases that, under the restricted control of a specific substrate, can generate free radicals. While the generation of nitric oxide (NO*) depends solely on the binding of L-arginine, NOS produces superoxide (O(2)*(-)) and hydrogen peroxide (H(2)O(2)) when the concentration of the substrate is low. Not surprisingly, effort has been put forth to understand the pathway by which NOS generates NO*, O(2)*(-) and H(2)O(2), including the role of substrate binding in determining the pathways by which free radicals are generated. By binding within the distal heme pocket near the sixth coordination position of the NOS heme iron, L-arginine alters the coordination properties of the heme iron that promotes formation of the perferryl complex NOS-[Fe(5+)=O](3+). This reactive iron intermediate has been shown to abstract a hydrogen atom from a carbon alpha to a heteroatom and generate carbon-centered free radicals. The ability of NOS to produce free radicals during enzymic cycling demonstrates that NOS-[Fe(5+)=O](3+) behaves like an analogous iron-oxo complex of cytochrome P-450 during aliphatic hydroxylation. The present review discusses the mechanism(s) by which NOS generates secondary free radicals that may initiate pathological events, along with the cell signaling properties of NO*, O(2)*(-) and H(2)O(2).     

A comparative study of neuronal and inducible nitric oxide synthases: generation of nitric oxide, superoxide, and hydrogen peroxide

Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (.NO), superoxide (O2.-), and hydrogen peroxide (H2O2). Since .NO has been implicated in many physiological processes, the importance of O2.- and H2O2 in regulating cell signaling by .NO cannot be overlooked. Before addressing these questions, we investigated the production of .NO, O2.-, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux of .NO from each isozyme supports differential biological activity. We found that the initial rate and sustained production of .NO was considerably greater for NOS 2 as compared to NOS 1. In the absence of L-arginine, however, NOS 1 generation of O2.- and H2O2 was found to be substantially greater than that measured for NOS 2. Differences between NOS 1 and NOS 2 production of .NO, O2.-, and H2O2 may define the specific physiologic function of each isozyme.     

Hemoglobins dioxygenate nitric oxide with high fidelity

Distantly related members of the hemoglobin (Hb) superfamily including red blood cell Hb, muscle myoglobin (Mb) and the microbial flavohemoglobin (flavoHb) dioxygenate nitric oxide (.NO). The reaction serves important roles in .NO metabolism and detoxification throughout the aerobic biosphere. Analysis of the stoichiometric product nitrate shows greater than 99% double O-atom incorporation from Hb(18)O(2), Mb(18)O(2) and flavoHb(18)O(2) demonstrating a conserved high fidelity .NO dioxygenation mechanism. Whereas, reactions of .NO with the structurally unrelated Turbo cornutus MbO(2) or free superoxide radical (-O.(2)) yield sub-stoichiometric nitrate showing low fidelity O-atom incorporation. These and other results support a .NO dioxygenation mechanism involving (1) rapid reaction of .NO with a Fe(III-)O.(2) intermediate to form Fe(III-)OONO and (2) rapid isomerization of the Fe(III-)OONO intermediate to form nitrate. A sub-microsecond isomerization event is hypothesized in which the O-O bond homolyzes to form a protein caged [Fe(IV)O .NO(2)] intermediate and ferryl oxygen attacks .NO(2) to form nitrate. Hb functions as a .NO dioxygenase by controlling O(2) binding and electrochemistry, guiding .NO diffusion and reaction, and shielding highly reactive intermediates from solvent water and biomolecules.     

Generation of nitric oxide by enzymatic oxidation of N-hydroxy-N-nitrosamines

The nitric oxide (N = O) free radical exhibits potent cytocidal, mutagenic and vasodilatory properties. We have examined the hypothesis that the hydroxynitrosamino functionality (see sequence in text), which occurs naturally in antineoplastic and antihypertensive agents, will directly generate N = O following peroxidatic 1-electron oxidation. Cupferron (see sequence in text) is indeed an excellent (k greater than 10(7) m-1 s-1) substrate for horseradish peroxidase. The products are N = O and nitrosobenzene (phi - N = O) which are generated and consumed as follows. First, cupferron is oxidized by the classical peroxidatic mechanism to form an unstable nitroxide free radical (see sequence in text) which then forms N = O and phi - N = O spontaneously (see sequence in text). The N = O then reacts with phi - N = O to reform cupferron (see sequence in text) or with the enzyme to generate the characteristic peroxidase--N = O chromophore. Simultaneously, in a competitive reaction with O2, the N = O is converted to NO-2 (4N = O + O2 + 2H2O------------4NO-2 + 4H+). The reactivity of hydroxynitrosamino compounds with horseradish peroxidase is in the order cupferron greater than hydroxynitrosaminomethane greater than alanosine. These model reactions, involving direct oxidation of the hydroxynitrosamino moiety, comprise a novel pathway for the biological production of N = O.     

Production and consumption of nitric oxide by three methanotrophic bacteria

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N(2)O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 x 10(-17) mol of NO cell(-1) day(-1), mostly after a culture became O(2) limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O(2), and required CH(4). Denitrification (methanol-supported N(2)O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd(1) and Cu nitrite reductases, NO reductase, and N(2)O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O(2) and nitrate availability occur.     

Fast dissociation of nitric oxide from ferrous Pseudomonas aeruginosa cd1 nitrite reductase. A novel outlook on the catalytic mechanism

The heme-containing periplasmic nitrite reductase (cd(1) NIR) is responsible for the production of nitric oxide (NO) in denitrifying bacterial species, among which are several animal and plant pathogens. Heme NIRs are homodimers, each subunit containing one covalently bound c-heme and one d(1)-heme. The reduction of nitrite to NO involves binding of nitrite to the reduced protein at the level of d(1)-heme, followed by dehydration of nitrite to yield NO and release of the latter. The crucial rate-limiting step in the catalytic mechanism is thought to be the release of NO from the d(1)-heme, which has been proposed, but never demonstrated experimentally, to occur when the iron is in the ferric form, given that the reduced NO-bound derivative was presumed to be very stable, as in other hemeproteins. We have measured for the first time the kinetics of NO binding and release from fully reduced cd(1) NIR, using the enzyme from Pseudomonas aeruginosa and its site-directed mutant H369A. Quite unexpectedly, we found that NO dissociation from the reduced d(1)-heme is very rapid, several orders of magnitude faster than that measured for b-type heme containing reduced hemeproteins. Because the rate of NO dissociation from reduced cd(1) NIR, measured in the present report, is faster than or comparable with the turnover number, contrary to expectations this event may well be on the catalytic cycle and not necessarily rate-limiting. This finding also provides a rationale for the presence in cd(1) NIR of the peculiar d(1)-heme cofactor, which has probably evolved to ensure fast product dissociation.     

Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM300 and reconstitution with native and synthetic heme d1.

Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.