Structural, redox, and mechanistic parameters for cysteine-sulfenic acid function in catalysis and regulation.

A primary objective of this review is to facilitate the application of the chemical and structural approaches that are currently being employed in the identification of Cys-SOH, as both transient intermediates and stable redox forms, in biochemical systems where these derivatives are suspected of playing key roles in redox catalysis or regulation. These range from high-resolution crystallographic analyses benefiting from recent technological advances in rapid data collection at cryogenic temperatures to 13C NMR investigations of [3-(13)C]Cys-labeled proteins and chemical modification protocols that can be integrated with both UV-visible and fluorescence spectroscopic as well as mass spectrometric (especially ESI, MALDI-TOF, and even FT ion-cyclotron-resonance) analyses. In summarizing the diversity of biological functions currently identified with Cys-SH reversible Cys-SOH redox cycles (Fig. 17), it should also be [figure: see text] emphasized that in at least one protein (nitrile hydratase) stable Cys-SOH and Cys-SO2H derivatives play important structural roles while also modulating the electronic properties of the iron center; in neither case is the Cys-SOH residue itself involved in reduction and oxidation. The somewhat incomplete structural descriptions of the oxidized Cys forms involved in redox regulation of some transcription factors (e.g., BPV-1 E2 protein and activator protein-1) indicate that there is ample room for the application of the types of investigations employed, for example, with NADH peroxidase and the AhpC peroxiredoxin, with a view toward defining the potential roles of Cys-SOH in these very important contexts of intracellular redox signaling. These advances will also build on the recent progress in defining sulfenic acid stabilization and properties in small molecule model systems, as evidenced in the work of Okazaki, Goto, and others. When viewed in the perspective of Allison's 1976 review on the subject of sulfenic acids in proteins, the reader will hopefully come to appreciate the conclusion that the concept of protein-sulfenic acids has now become a very well-defined and established principle of biochemistry, with current efforts in this and other laboratories being directed to bring about still more detailed understanding of Cys-SOH function in both redox and nonredox modes of enzyme catalysis and regulation of protein function.

Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes.

An understanding of how the heme-deficient gram-positive bacterium Streptococcus pyogenes establishes infections in O(2)-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic acid bacteria which directly catalyzes the four-electron reduction of O(2) to H(2)O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O(2) conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O(2) conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O(2) tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H(2)O(2) in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H(2)O(2) and rescued the growth defect of the mutants under high-O(2) conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O(2) with carbohydrate limitation.

Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation.

While it has been known for more than 20 years that unusually stable cysteine-sulfenic acid (Cys-SOH) derivatives can be introduced in selected proteins by mild oxidation, only recently have chemical and crystallographic evidence for functional Cys-SOH been presented with native proteins such as NADH peroxidase and NADH oxidase, nitrile hydratase, and the hORF6 and AhpC peroxiredoxins. In addition, Cys-SOH forms of protein tyrosine phosphatases and glutathione reductase have been suggested to play key roles in the reversible inhibition of these enzymes during tyrosine phosphorylation-dependent signal transduction events and nitrosative stress, respectively. Substantial chemical data have also been presented which implicate Cys-SOH in redox regulation of transcription factors such as Fos and Jun (activator protein-1) and bovine papillomavirus-1 E2 protein. Functionally, the Cys-SOHs in NADH peroxidase, NADH oxidase, and the peroxiredoxins serve as either catalytically essential redox centers or transient intermediates during peroxide reduction. In nitrile hydratase, the active-site Cys-SOH functions in both iron coordination and NO binding but does not play any catalytic redox role. In Fos and Jun and the E2 protein, on the other hand, a key Cys-SH serves as a sensor for intracellular redox status; reversible oxidation to Cys-SOH as proposed inhibits the corresponding DNA binding activity. These functional Cys-SOHs have roles in diverse cellular processes, including signal transduction, oxygen metabolism and the oxidative stress response, and transcriptional regulation, as well as in the industrial production of acrylamide, and their detailed analyses are beginning to provide the chemical foundation necessary for understanding protein-SOH stabilization and function.

Equilibrium analyses of the active-site asymmetry in enterococcal NADH oxidase: role of the cysteine-sulfenic acid redox center.

Recent studies [Mallett, T. C., and Claiborne, A. (1998) Biochemistry 37, 8790-8802] of the O2 reactivity of C42S NADH oxidase (O2 --> H2O2) revealed an asymmetric mechanism in which the two FADH2.NAD+ per reduced dimer display kinetic inequivalence. In this report we provide evidence indicating that the fully active, recombinant wild-type oxidase (O2 --> 2H2O) displays thermodynamic inequivalence between the two active sites per dimer. Using NADPH to generate the free reduced wild-type enzyme (EH2'/EH4), we have shown that NAD+ titrations lead to differential behavior as only one FADH2 per dimer binds NAD+ tightly to give the charge-transfer complex. The second FADH2, in contrast, transfers its electrons to the single Cys42-sulfenic acid (Cys42-SOH) redox center, which remains oxidized during the reductive titration. Titrations of the reduced NADH oxidase with oxidized 3-acetylpyridine and 3-aminopyridine adenine dinucleotides further support the conclusion that the two FADH2 per dimer in wild-type enzyme can be described as distinct "charge-transfer" and "electron-transfer" sites, with the latter site giving rise to either intramolecular (Cys42-SOH) or bimolecular (pyridine nucleotide) reduction. The reduced C42S mutant is not capable of intramolecular electron transfer on binding pyridine nucleotides, thus confirming that the Cys42-SOH center is in fact the source of the redox asymmetry observed with wild-type oxidase. These observations on the role of Cys42-SOH in the expression of thermodynamic inequivalence as observed in wild-type NADH oxidase complement the previously described kinetic inequivalence of the C42S mutant; taken together, these results provide the overlapping framework for an alternating sites cooperativity model of oxidase action.

The soluble alpha-glycerophosphate oxidase from Enterococcus casseliflavus. Sequence homology with the membrane-associated dehydrogenase and kinetic analysis of the recombinant enzyme.

The soluble flavoprotein alpha-glycerophosphate oxidase from Enterococcus casseliflavus catalyzes the oxidation of a "non-activated" secondary alcohol, in contrast to the flavin-dependent alpha-hydroxy- and alpha-amino acid oxidases. Surprisingly, the alpha-glycerophosphate oxidase sequence is 43% identical to that of the membrane-associated alpha-glycerophosphate dehydrogenase from Bacillus subtilis; only low levels of identity (17-22%) result from comparisons with other FAD-dependent oxidases. The recombinant alpha-glycerophosphate oxidase is fully active and stabilizes a flavin N(5)-sulfite adduct, but only small amounts of intermediate flavin semiquinone are observed during reductive titrations. Direct determination of the redox potential for the FAD/FADH2 couple yields a value of -118 mV; the protein environment raises the flavin potential by 100 mV in order to provide for a productive interaction with the reducing substrate. Steady-state kinetic analysis, using the enzyme-monitored turnover method, indicates that a ping-pong mechanism applies and also allows the determination of the corresponding kinetic constants. In addition, stopped-flow studies of the reductive half-reaction provide for the measurement of the dissociation constant for the enzyme. alpha-glycerophosphate complex and the rate constant for reduction of the enzyme flavin. These and other results demonstrate that this enzyme offers a very promising paradigm for examining the protein determinants for flavin reactivity and mechanism in the energy-yielding metabolism of alpha-glycerophosphate.

Oxygen reactivity of an NADH oxidase C42S mutant: evidence for a C(4a)-peroxyflavin intermediate and a rate-limiting conformational change.

The flavoprotein NADH oxidase (O2 --> 2H2O) from Enterococcus faecalis 10C1 contains a cysteinyl redox center, in addition to FAD. We have proposed a cysteine-sulfenic acid (Cys-SOH) structure for the oxidized form of Cys42; the presence of this redox center is consistent with the stoichiometries reported for earlier reductive titrations of wild-type oxidase, and we have proposed that Cys42-SH plays a key role in the overall four-electron reduction of O2 --> 2H2O. To test these proposals, we provide in this report an analysis of the oxidative half-reaction of an oxidase mutant in which Cys42 is replaced by Ser. NADH titrations lead to direct flavin reduction with 1.05 equiv of NADH/FAD and give rise to the formation of a very stable E-FADH2.NAD+ complex. Kinetic analyses indicate that this species is catalytically competent, and its reactivity with O2 has been analyzed in detail by stopped-flow spectrophotometry using both single-wavelength and diode-array modes of data acquisition. The combined results of this analysis demonstrate that replacement of Cys42 with Ser provides for an altered O2 reduction stoichiometry in which H2O2, not 2H2O, is the product. The two subunits of the reduced enzyme.NAD+ complex react with O2 in an asymmetric mechanism, consistent with an alternating sites cooperativity model such as that proposed [Miller, S. M., Massey, V., Williams, C. H., Jr., Ballou, D. P., and Walsh, C. T. (1991) Biochemistry 30, 2600-2612] for mercuric reductase. An FAD C(4a)-hydroperoxide is identified as the primary oxygenated intermediate in reoxidation of the complex, but the reaction of O2 with the complementary subunit does not proceed until full reoxidation has occurred at the primary subunit. To our knowledge, this is the first report of a C(4a)-peroxyflavin intermediate outside the flavoprotein monooxygenase class.

Purification and characterization of glucose-6-phosphate dehydrogenase from Cryptococcus neoformans: identification as "nothing dehydrogenase".

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Cryptococcus neoformans, a basidiomyceteous yeast that is an opportunistic pathogen of AIDS patients. The enzyme had a subunit molecular weight of 5 x 10(4), a specific activity of 50 units mg-1, and Km values for NADP and glucose-6-phosphate of 1.6 and 24 microM, respectively. The enzyme catalyzed the dehydrogenation of glucose, in the presence of dimethylsulfoxide, with Km of 5 mM and Vmax 10% of that with glucose-6-phosphate. pH profiles indicated the presence of a group with pKa of 6.6 that is involved in catalysis, and groups with pKas of about 8.8 that are involved in binding of NADP and glucose-6-phosphate. The enzyme was inhibited by NADPH, competitive versus NADP, with Ki of 1 microM, and by zinc ion, competitive versus glucose-6-phosphate, with Ki of 2 microM. Crude enzyme extract catalyzed an appreciable rate of reduction of NADP in the absence of added substrate, a "nothing dehydrogenase" activity. This activity was shown to be due to the presence of glucose-6-phosphate in the crude extract. It was calculated that cells of C. neoformans contain about 25 mumol of glucose-6-phosphate per gram, wet weight.