REDOX TITRATIONS OF CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM

被引:36
作者
SHIN, W [1 ]
STAFFORD, PR [1 ]
LINDAHL, PA [1 ]
机构
[1] TEXAS A&M UNIV SYST,DEPT CHEM,COLLEGE STN,TX 77843
关键词
D O I
10.1021/bi00141a007
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
Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the g(av) = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983. The g(av) = 1.82 species either is not an iron-sulfur cluster or is one with unusual optical and redox properties. Thionin in large molar amounts slowly inactivated the enzyme by an oxidation process. Exposure of 100 equiv/mol of thionin to CODH for 1 week completely inactivated the enzyme. These so-called group III oxidations are not involved in the catalytic mechanism of the enzyme.
引用
收藏
页码:6003 / 6011
页数:9
相关论文
共 34 条
[11]  
Lancaster J.R., 1988, BIOINORGANIC CHEM NI
[12]  
Lindahl P.A., 1990, J CLUST SCI, V1, P29
[13]  
LINDAHL PA, 1990, J BIOL CHEM, V265, P3880
[14]  
LINDAHL PA, 1988, J BIOL CHEM, V263, P19412
[15]  
LINDAHL PA, 1990, J BIOL CHEM, V265, P3873
[16]  
LU WP, 1990, J BIOL CHEM, V265, P3124
[17]   DEVELOPMENT OF A MINIMALLY DEFINED MEDIUM FOR THE ACETOGEN CLOSTRIDIUM-THERMOACETICUM [J].
LUNDIE, LL ;
DRAKE, HL .
JOURNAL OF BACTERIOLOGY, 1984, 159 (02) :700-703
[18]  
MAYHEW SG, 1969, J BIOL CHEM, V244, P2830
[19]   IRON-SULFUR PROTEINS - STRUCTURE AND FUNCTION [J].
ORMEJOHN.WH .
ANNUAL REVIEW OF BIOCHEMISTRY, 1973, 42 :159-204
[20]   SIMPLE RAPID BIURET METHOD FOR ESTIMATION OF PROTEIN IN SAMPLES CONTAINING THIOLS [J].
PELLEY, JW ;
GARNER, CW ;
LITTLE, GH .
ANALYTICAL BIOCHEMISTRY, 1978, 86 (01) :341-343