Two-pronged survival strategy for the major cystic fibrosis pathogen, Pseudomonas aeruginosa, lacking the capacity to degrade nitric oxide during anaerobic respiration.

Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated approximately 13.6 microM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50- and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S](2+) cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O(2)-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.

Aromatic ring cleavage by homoprotocatechuate 2,3-dioxygenase: role of His200 in the kinetics of interconversion of reaction cycle intermediates.

Homoprotocatechuate 2,3-dioxygenase (WT 2,3-HPCD) isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the aromatic ring of the substrate (HPCA). Here, the conserved active site residue His200 is changed to Gln, Glu, Ala, Asn, and Phe, and the reactions of the mutant enzymes are probed using steady-state and transient kinetic techniques. Each mutant catalyzes ring cleavage of HPCA to yield the normal product. H200Q and H200N retain 30-40% of the WT 2,3-HPCD activity at 24 degrees C, but the other mutants reduce the k(cat) to less than 9% of normal. The origin of the reduced activity is unlikely to be the substrate binding phase of the catalytic cycle, because the multistep anaerobic binding reaction of the chromophoric substrate 4-nitrocatechol (4NC) is shown to proceed with rate constants similar to those observed for WT 2,3-HPCD. In contrast, the rate constants of several steps in the multistep O(2) binding/insertion and product release half of the reaction cycle are substantially slowed, in particular the steps in which activated oxygen attacks the organic substrate and in which product is released. In the case of the H200N mutant, the product of 4NC oxidation is not the usual ring cleavage product, but rather the 4NC quinone. These results suggest that the main role of His200 is in facilitating the steps in the second half of the reaction cycle. The decreased rate constants for the O(2) insertion steps in the catalytic cycles of the mutant enzymes allow the oxygen adduct of an extradiol dioxygenase to be detected for the first time.

Single-turnover kinetics of homoprotocatechuate 2,3-dioxygenase.

Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the substrate aromatic ring. In contrast to other members of the ring cleaving dioxygenase family, the transient kinetics of the extradiol dioxygenase catalytic cycle have been difficult to study because the iron is nearly colorless and EPR silent. Here, it is shown that the reaction cycle kinetics can be monitored by utilizing the alternative substrate 4-nitrocatechol (4NC), which is also cleaved in the proximal extradiol position. Changes in the optical spectrum of 4NC occurring as a result of ionization, environmental changes, and ring cleavage allow both the substrate binding and product formation phases of the reaction to be studied. It is shown that substrate binding occurs in a four-step process probably involving binding to two ionization states of the enzyme at different rates. Following an initial rapid binding of the monoanionic 4NC in the active site, slower binding to the Fe(II) and conversion to the dianionic form occur. The bound dianionic 4NC reacts rapidly with O(2) in four additional steps, apparently occurring in sequence. On the basis of the optical properties of the intermediates, these steps are hypothesized to be O(2) binding to the iron, isomerization of the resulting complex, ring opening, and product release. The natural substrate appears to form the same intermediates but with much larger rate constants. These are the first transient intermediates to be reported for an extradiol dioxygenase reaction.

Conversion of extradiol aromatic ring-cleaving homoprotocatechuate 2,3-dioxygenase into an intradiol cleaving enzyme.

The intra- and extradiol subfamilies of catechol-adduct ring-cleaving dioxygenases each exhibit nearly absolute fidelity for the ring cleavage position. This is often attributed to the fact that the oxygen activation mechanism of intradiol dioxygenases utilizes Fe3+ while that of the extradiol enzymes employs Fe2+, but the subfamilies also differ in primary sequence, structural fold, iron ligands, and second sphere active site amino acid residues. Here, we examine the effects of the second sphere residue H200 in the active site of homoprotocatechuate 2,3-dioxygenase (2,3-HPCD), an extradiol-cleaving enzyme. It is shown that the H200F mutant enzyme catalyzes extradiol cleavage of the normal substrate, homoprotocatechuate (HPCA), but intradiol cleavage of the alternative substrate 2,3-dihydroxybenzoate (2,3-DHB) while in the Fe2+ oxidation state. Wild-type 2,3-HPCD catalyzes extradiol cleavage of both substrates. This is the first report of intradiol cleavage by an extradiol dioxygenase. It suggests that intradiol cleavage can occur with the iron in the Fe2+ state, with the iron ligand set characteristic of extradiol dioxygenases, and through a mechanism in which oxygen is activated by binding to the iron rather than directly attacking the substrate as in true intradiol dioxygenases. This indicates that substrate binding geometry and acid/base chemistry of second sphere residues play important roles in determining the course of the dioxygenase reaction.