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The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O2 and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O–O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV–visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pKa values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pKa of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid–base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.

Flavin-binding LOV domains are broadly conserved in plants, fungi, archaea, and bacteria. These ≈100 residue photosensory modules are generally encoded within larger, multi-domain proteins that control a range of blue light-dependent physiologies. The bacterium Caulobacter crescentus encodes a soluble LOV-histidine kinase, LovK, that regulates the adhesive properties of the cell. Full-length LovK is dimeric as are a series of systematically truncated LovK constructs containing only the N-terminal LOV sensory domain. Non-conserved sequence flanking the LOV domain functions to tune the signaling lifetime of the protein. Size exclusion chromatography and small angle X-ray scattering (SAXS) demonstrate that the LOV sensor domain does not undergo a large conformational change in response to photon absorption. However, limited proteolysis identifies a sequence flanking the C-terminus of the LOV domain as a site of light-induced change in protein conformation/dynamics. Based on SAXS envelope reconstruction and bioinformatic prediction, we propose this dynamic region of structure is an extended C-terminal coiled-coil that links the LOV domain to the histidine kinase domain. To test the hypothesis that LOV domain signaling is affected by cellular redox state in addition to light, we measured the reduction potential of the LovK FMN cofactor. The measured potential of −258 mV is congruent with the redox potential of gram-negative cytoplasm during logarithmic growth (−260 to −280 mV). Thus a fraction of LovK in the cytosol may be in the reduced state under typical growth conditions. Chemical reduction of the FMN cofactor of LovK attenuates light-dependent ATPase activity of the protein in vitro, demonstrating that LovK can function as a conditional photosensor that is regulated by the oxidative state of the cellular environment.Cellular adaptation requires protein domains that can perceive physical or chemical changes in the environment, and transduce detection of such events to downstream effectors. Flavinbinding light-, oxygen-, or voltage (LOV) domains, which were first identified in the phototropin (phot) plant photoreceptor family (1), are well-characterized protein † E.B.P was supported by National Institutes of Health Training Grant 5T32GM007183-34. S.C. acknowledges support for this project from NIH grant 1R01GM087353-2, the Arnold and Mabel Beckman Foundation (BYI), and the Mallinckrodt Foundation. Advanced Photon Source is supported by the DOE Office of Basic Energy Sciences (Contract No. DE-AC02-06CH11357). BioCAT is an NIHsupported Research Center (RR-08630). C.A.M. and B.A.P. are supported by NIH grant 2R01GM061087-8. * Corresponding Author: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, GCIS W138, Chicago, IL 60637, Phone: (773) 834-1926, Fax: (773) 702-0439, scrosson@uchicago.edu. Supporting Information AvailableAn alignment of (A) N-terminal and (B) C-terminal LOV domain flanking sequence not presented in Figure 1 is available free of charge via ...

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometrybased analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycopro-

Many flavoenzymes – oxidases and monooxygenases – react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused on the importance of a positive charge near N5 of the reduced flavin. Fructosamine oxidase has a lysine near N5 of its flavin. We measured a rate constant of 1.6 × 105 M−1s−1 for its reaction with oxygen. The Lys276Met mutant reacted with a rate constant of 291 M−1s−1, suggesting an important role for this lysine in oxygen activation. The dihydroorotate dehydrogenases from E. coli and L. lactis also have a lysine near N5 of the flavin. They react with O2 with rate constants of 6.2 × 104 M−1s−1 and 3.0 × 103 M−1s−1, respectively. The Lys66Met and Lys43Met mutant enzymes react with rate constants that are nearly the same as the wild-type enzymes, demonstrating that simply placing a positive charge near N5 of the flavin does not guarantee increased oxygen reactivity. Our results show that the lysine near N5 does not exert an effect without an appropriate context; evolution did not find only one mechanism for activating the reaction of flavins with O2.