Vitreoscilla Hemoglobin

Ramandeep,; Hwang, Kwang Woo; Raje, Manoj; Kim, Kyungjin; Stark, Benjamin C.; Dikshit, Kanak L.; Webster, D. R.

doi:10.1074/jbc.m009808200

Cited by 136 publications

(30 citation statements)

References 40 publications

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“…These questions might be answered using site-directed mutants; a nonoxygen binding mutant of VHb could be used to test the former, for example, and exterior surface mutants the latter. In the previously reported binding studies (13), VHb bound equally well to both E. coli and Vitreoscilla membranes, which is supported by the results of the two-hybrid assay of the current study. An interaction between cytochrome bo and VHb was suggested by Tsai et al (3).…”

Section: Construction Of Peg202::vgb and Pjg4-5::cyo Bo Ubiquinolsupporting

confidence: 75%

“…Using immunogold labeling of VHb in E. coli and Vitreoscilla, the cellular localization of VHb has been recently determined to be in the cytoplasm, concentrated in the outer perimeter near the cell membrane (13). The same study showed that VHb bound to bacterial (E. coli and Vitreoscilla) respiratory membranes, which would account for its localization.…”

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confidence: 84%

“…To attempt to identify the binding sites in the membranes, the binding of VHb to Vitreoscilla cytochrome bo in synthetic membrane vesicles was tested, and a K d of 6.2 M was determined. The observation that exogenously added VHb stimulated the ubiquinol oxidase activity of both the respiratory membranes and the cytochrome bo proteoliposomes, especially under hypoxic conditions (13), is evidence that the interaction between VHb and cytochrome bo is of physiological importance. To obtain independent verification for this interaction and to attempt to identify which subunit or subunits in the cytochrome are the actual binding targets, we further examined this interaction using the yeast two-hybrid system (14) with VHb as the bait and individual subunits and parts of subunits of Vitreoscilla cytochrome bo as prey.…”

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confidence: 99%

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Vitreoscilla Hemoglobin Binds to Subunit I of Cytochrome bo Ubiquinol Oxidases

Park

Kim

Howard

et al. 2002

Journal of Biological Chemistry

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106

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The bacterium, Vitreoscilla, can induce the synthesis of a homodimeric hemoglobin under hypoxic conditions. Expression of VHb in heterologous bacteria often enhances growth and increases yields of recombinant proteins and production of antibiotics, especially under oxygen-limiting conditions. There is evidence that VHb interacts with bacterial respiratory membranes and cytochrome bo proteoliposomes. We have examined whether there are binding sites for VHb on the cytochrome, using the yeast two-hybrid system with VHb as the bait and testing every Vitreoscilla cytochrome bo subunit as well as the soluble domains of subunits I and II. A significant interaction was observed only between VHb and intact subunit I. We further examined whether there are binding sites for VHb on cytochrome bo from Escherichia coli and Pseudomonas aeruginosa, two organisms in which stimulatory effects of VHb have been observed. Again, in both cases a significant interaction was observed only between VHb and subunit I. Because subunit I contains the binuclear center where oxygen is reduced to water, these data support the function proposed for VHb of providing oxygen directly to the terminal oxidase; it may also explain its positive effects in Vitreoscilla as well as in heterologous organisms.

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Section: Construction Of Peg202::vgb and Pjg4-5::cyo Bo Ubiquinolsupporting

confidence: 75%

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confidence: 84%

mentioning

confidence: 99%

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Vitreoscilla Hemoglobin Binds to Subunit I of Cytochrome bo Ubiquinol Oxidases

Park

Kim

Howard

et al. 2002

Journal of Biological Chemistry

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106

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“…An inspection of the absorption spectrum of the unliganded derivative is consistent with the presence of a pentacoordinated heme adduct. It is worth noting that despite the contribution attributed to the flavin absorption (shoulders at 470 and 490 nm with a broad tail toward the visible region), the peak at 645 nm and the broad Soret band characterized by a low molar absorptivity and centered at ϳ403 nm are clearly identified as typical pentacoordinate high spin marker bands in which the proximal histidine is the fifth ligand (22). The spectrum of the fully liganded species is typical of hexacoordinate low spin adducts observed in all other hemoglobins and myoglobins.…”

Section: Fhp) and Thrmentioning

confidence: 99%

The X-ray Structure of Ferric Escherichia coliFlavohemoglobin Reveals an Unexpected Geometry of the Distal Heme Pocket

Ilari¹,

Bonamore²,

Farina³

et al. 2002

Journal of Biological Chemistry

130

137

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The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 Å and refined to an R-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.Flavohemoglobins are oxygen-binding proteins composed of a typical globin domain containing a B-type heme fused with a ferredoxin reductase-like FAD-and NAD-binding domain. Together with single domain bacterial hemoglobins and six-helices containing "truncated hemoglobins," flavohemoglobins are part of a vast family of "hemoglobin-like" proteins whose functions are still elusive. Because of the identification of flavohemoglobin genes in a wide variety of prokaryotic and eukaryotic microorganisms, a crescendo of experimental observations has revealed that these proteins possess unique structural and functional properties unrelated to those of hemoproteins involved in oxygen transport and storage (1).Escherichia coli flavohemoglobin (HMP) 1 is certainly the most extensively studied protein within the bacterial hemoglobin family. At present, the most credited physiological function of HMP has been inferred from the observation that protein expression in the bacterial cell is enhanced in the presence of nitric oxide releasers in the culture medium (2-4). Accordingly, HMP has been shown to be able to catalyze the oxidation of free nitric oxide to nitrate both in vivo and in vitro in the presence of oxygen and NADH (5). In this framework, HMP is thought to be involved in the response of the bacterial cell to the potentially harmful role of free nitric oxide in giving rise to nitrosative stress. Nevertheless, although the nitric-oxide dioxygenase activity stands as the major functional hypothesis for HMP, the molecular mechanism and the structural determinants at the basis of this enzymic function remain poorly understood. In particular, it has not been established as yet how the amino acid side chains in the distal heme pocket affect...

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“…The VHb gene is a hemoglobin-like gene present in the chromosome of the aerobic Gram-negative bacterium, Vitreoscilla sp., whose protein plays an important role in oxygen transfer [11,17,22,25]. Heterologous expressions of the VHb gene in several microorganisms, including E. coli, stimulates ATP generation by promoting efficient oxygen transfer in an oxygen-limited environment, leading to enhanced cell growth and protein synthesis [2,5,14,15,27]. A VHb gene cloned in a fungal expression vector, pBARGPE1, containing a selection marker of an Ignite/basta-resistance (bar) gene [12] was kindly provided by Prof. G. T. Chun of Kangwon University, Korea.…”

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confidence: 99%