Structural features of covalently cross-linked hydroxylase and reductase proteins of soluble methane monooxygenase as revealed by mass spectrometric analysis

被引:16
作者
Kopp, DA
Berg, EA
Costello, CE
Lippard, SJ [1 ]
机构
[1] MIT, Dept Chem, Cambridge, MA 02139 USA
[2] Boston Univ, Sch Med, Mass Spectrometry Resource, Boston, MA 02118 USA
关键词
D O I
10.1074/jbc.M301581200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Soluble methane monooxygenase requires complexes between its three component proteins for efficient catalysis. The hydroxylase (MMOH) must bind both to the reductase (MMOR) and to the regulatory protein (MMOB) to facilitate oxidation of methane to methanol. Although structures of MMOH, MMOB, and one domain of MMOR have been determined, less geometric information is available for the complexes. To address this deficiency, MMOH and MMOR were cross-linked by a carbodiimide reagent and analyzed by specific proteolysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and capillary high performance liquid chromatography mass spectrometry. Tandem mass spectra conclusively identified two amine-to-carboxylate cross-linked sites involving the alpha subunit of MMOH and the [2Fe-2S] domain of MMOR (MMOR-Fd). In particular, the N terminus of the MMOH alpha subunit forms cross-links to the side chains of MMOR-Fd residues Glu-56 and Glu-91. These Glu residues are close to one another on the surface of MMOR-Fd and >25 Angstrom from the [2Fe-2S] cluster. Because the N terminus of the alpha subunit of MMOH was not located in the crystal structure of MMOH, a detailed structural model of the complex based on the cross-link was precluded; however, a previously proposed binding site for MMOR on MMOH could be ruled out. Based on the cross-linking results, a MMOR E56Q/E91Q double mutant was generated. The mutant retains >80% of MMOR NADH oxidase activity but reduces sMMO activity to similar to65% of the level supported by the wild type reductase. Cross-linking to MMOH was diminished but not abolished in the double mutant, indicating that other residues of MMOR also form cross-links to MMOH.
引用
收藏
页码:20939 / 20945
页数:7
相关论文
共 39 条
[1]   Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerise holoenzyme [J].
Alley, SC ;
Trakselis, MA ;
Mayer, MU ;
Ishmael, FT ;
Jones, AD ;
Benkovic, SJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (42) :39340-39349
[2]   Positively charged amino acids are essential for electron transfer and protein-protein interactions in the soluble methane monooxygenase complex from Methylococcus capsulatus (Bath) [J].
Balendra, S ;
Lesieur, C ;
Smith, TJ ;
Dalton, H .
BIOCHEMISTRY, 2002, 41 (08) :2571-2579
[3]   Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) [J].
Blazyk, JL ;
Lippard, SJ .
BIOCHEMISTRY, 2002, 41 (52) :15780-15794
[4]   The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry [J].
Buzy, A ;
Millar, AL ;
Legros, V ;
Wilkins, PC ;
Dalton, H ;
Jennings, KR .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1998, 254 (03) :602-609
[5]   Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent [J].
Cai, K ;
Itoh, Y ;
Khorana, FC .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (09) :4877-4882
[6]   Solution structure of component B from methane monooxygenase derived through heteronuclear NMR and molecular modeling [J].
Chang, SL ;
Wallar, BJ ;
Lipscomb, JD ;
Mayo, KH .
BIOCHEMISTRY, 1999, 38 (18) :5799-5812
[7]   Residues in Methylosinus trichosporium OB3b methane monooxygenase component B involved in molecular interactions with reduced- and oxidized-hydroxylase component:: A role for the N-terminus [J].
Chang, SL ;
Wallar, BJ ;
Lipscomb, JD ;
Mayo, KH .
BIOCHEMISTRY, 2001, 40 (32) :9539-9551
[8]   PHTHALATE DIOXYGENASE REDUCTASE - A MODULAR STRUCTURE FOR ELECTRON-TRANSFER FROM PYRIDINE-NUCLEOTIDES TO [2FE-2S] [J].
CORRELL, CC ;
BATIE, CJ ;
BALLOU, DP ;
LUDWIG, ML .
SCIENCE, 1992, 258 (5088) :1604-1610
[9]   Sequencing and analysis of the Methylococcus capsulatus (Bath) soluble methane monooxygenase genes [J].
Coufal, DE ;
Blazyk, JL ;
Whittington, DA ;
Wu, WW ;
Rosenzweig, AC ;
Lippard, SJ .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 2000, 267 (08) :2174-2185
[10]  
Elango N, 1997, PROTEIN SCI, V6, P556