Adrenal ascorbate-dependent cytochrome (cyt.) b561 is a transmembrane electron transporter required for synthesis of catecholamine and some peptide hormone and is the prototype of an expanding and insufficiently characterized cyt. b561 protein family;other mammalian family members have been linked to intestinal iron transport and tumor suppression. Our overall goal is to characterize the mechanism of adrenal b561 and to use information about this cytochrome to understand the structure and function of other b561 family members.
The specific aims of this project are: 1) Define the structural kernel of adrenal cyt. b561, determine its relationship to the membrane bilayer, and evaluate the kernel's generality among mammalian cyt. b561 family members;2) Characterize the interaction of adrenal cyt. b561 with its natural reductant, ascorbate, and its natural oxidant, semidehydroascorbate;and 3) Determine the path of electron flow via adrenal cyt. b561 and its regulation. Planned experiments will be conducted on the native mammalian protein and recombinant wild type and mutant forms of the adrenal cytochrome and other mammalian cyt. b561 family members in prokaryotic and eukaryotic systems that we have developed. We will characterize the topological, structural, and kinetic properties of these recombinant proteins and purified endogenous adrenal cyt b561 using biochemical and biophysical techniques. Understanding how adrenal cyt. b561 functions is a fundamental part of understanding the physiology and pathology of epinephrine and norepinephrine and of some peptide hormones in humans. Defining similarities and differences between adrenal cyt. b561 and recently-discovered human cyt. b561 family members will accelerate characterization of the physiological and pathological roles of the newer cyt. b561s.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080575-03
Application #
7585163
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2007-04-02
Project End
2011-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
3
Fiscal Year
2009
Total Cost
$283,991
Indirect Cost
Name
Rice University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
050299031
City
Houston
State
TX
Country
United States
Zip Code
77005
Jancura, Daniel; Stanicova, Jana; Palmer, Graham et al. (2014) How hydrogen peroxide is metabolized by oxidized cytochrome c oxidase. Biochemistry 53:3564-75
da Silva, Giordano F Z; Shinkarev, Vladimir P; Kamensky, Yury A et al. (2012) Spectroscopic evidence of the role of an axial ligand histidinate in the mechanism of adrenal cytochrome b(561). Biochemistry 51:8730-42
Liu, Wen; Wu, Gang; Tsai, Ah-Lim et al. (2011) High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system. Protein Expr Purif 79:115-21
Liu, Wen; da Silva, Giordano F Z; Wu, Gang et al. (2011) Functional and structural roles of residues in the third extramembrane segment of adrenal cytochrome b561. Biochemistry 50:3149-60
Parul, Dzmitry; Palmer, Graham; Fabian, Marian (2010) Ligand trapping by cytochrome c oxidase: implications for gating at the catalytic center. J Biol Chem 285:4536-43
Sato, Hideaki; Sugishima, Masakazu; Sakamoto, Hiroshi et al. (2009) Crystal structure of rat haem oxygenase-1 in complex with ferrous verdohaem: presence of a hydrogen-bond network on the distal side. Biochem J 419:339-45
Liu, Wen; Rogge, Corina E; da Silva, Giordano F Z et al. (2008) His92 and His110 selectively affect different heme centers of adrenal cytochrome b(561). Biochim Biophys Acta 1777:1218-28
Higashimoto, Yuichiro; Sugishima, Masakazu; Sato, Hideaki et al. (2008) Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase. Biochem Biophys Res Commun 367:852-8
Kamensky, Yury; Liu, Wen; Tsai, Ah-Lim et al. (2007) Axial ligation and stoichiometry of heme centers in adrenal cytochrome b561. Biochemistry 46:8647-58
Sato, Hideaki; Higashimoto, Yuichiro; Sakamoto, Hiroshi et al. (2007) Electrochemical reduction of ferrous alpha-verdoheme in complex with heme oxygenase-1. J Inorg Biochem 101:1394-9