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FINAL TECHNICAL REPORT 

NASA GRANT NAGW-342 i 

THIOL BIOCHEMISTRY OF PROKARYOTES 

Principal Investigator: Robert C. Fahey J 

Department of Chemistry D-006 

University of California, San Diego 

La Jolla CA 92093 

(619) 534-2163, 534-4821 

Grantee Institution: University of California, San Diego 

Period Covered: June 7, 1982 through December 31, 1986 

(bASA-CR-780257) THICL BICCEE@ISIBP OP BE?-1 1579 

ESGKABYCIES Einal Technical Eeprt, 7 Jun, 

IS82 - 31 Dec. 19€6 (Califcrcia Oniv., San 

Liego, La Jolla.) 5 p CSCL 06P Unclas 

G3/52 43816 

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BRIEF SUMMARY OF PROJECT 

Low molecular weight thiols play a key role in cellular defenses against 

oxygen toxicity. 

shown that it is absent in many prokaryotes. 

have existed in prokaryotes prior to the evolution of cyanobacteria and 

accumulation of oxygen in the atmosphere, it might be expected that 

evolutionarily distinct groups of bacteria evolved mechanisms of protection 

against oxygen toxicity based upon different low molecular weight thiols. The 

long term goal of this research is to delineate the differences in the thiol 

biochemistry of prokaryotes and to understand how these differences evolved in 

response to the shift from anaerobic to aerobic metabolism. 

this proposed research are: (1) to expand the methods of thiol analysis 

developed in our laboratory to measure all known biological thiols and to 

include analyses for thioesters, disufides , and dithiol proteins (thioredoxin) ; 

(2) to apply these methods to establish the main low molecular weight thiols 

produced by all major groups of prokaryotes; (3) to establish the structures of 

any novel thiols which appear to have wide distribution; (4) to elaborate the 

occurence of key enzymes, especially disulfide reductases, involved in the 

metabolism of these thiols. 

Glutathione is the thiol used in higher organisms but we have 

Since substantial diversity must 

Specific aims of 

With respect to the first specific aim, the methodology based upon 

fluorescent labeling with monobromobimane for analysis of thiols and thiol 

derivatives has been greatly extended and now includes ion pairig HPLC 

techniques which permit analysis of low molecular weight thiols that are highly 

anionic in character, such as coenzyme A, as well as cationic thiols (Pl,P7). 

Methods for specific analysis of the thiol components of thioesters and 

disulfides proved more difficult to develop than expected owing to the fact that 

conventional methods used to cleave disulfides also cleave thioesters but 

selective cleavage based upon hydroxylamine led to a successful method (P4). 

An analysis for thioredoxin was also developed based upon fluorescent labeling 

of the two cysteine residues and HPK analysis after a heat treatment and 

desalting procedure (P5). To facilitate isolation and identification of thiols 

of unknown or uncertain structure from biological systems a rapid and efficient 

method for purification of thiols from biological extracts was developed based 

upon thiol affinity chromatography and preparative HPLC (P8). 

In accomplishing specific aims (2) and (3) we have shown that glutathione 

Since the purple bacteria are 

is absent in all strictly anaerobic bacteria studied and is produced only by the 

purple bacteria and the cyanobacteria (Pl, ~6). 

the group most closely related to mitochondria and the cyanobacteria are related 

to chloroplasts, this suggests that incorporation of glutathione metabolism into 

eucaryotes could have occurred during the endosymbiosis giving rise to 

mitochondria and chloroplasts. This hypothesis is supported by the finding that 

Entamoeba histolytica, a eucaryote lacking both mitochondria and chloroplasts, 

does not produce glutathione or the enzymes of glutathione metabolism (P2). To 

further understand mechanisms of protection against oxygen toxicity it was 

important to ascertain what thiols are used by aerobic bacteria which lack GSH. 

The Gram positive bacteria and the radiobacteria were found to produee coenzyme 

A in unusually large amounts (Pl) whereas, among the archaebacteria, the 

halobacteria were shown to produce half of the glutathione molecule, 

1-glutangrlcysteine, as the major intracellular thiol (P3). 

unidentified thiols were also detected, but few occurred at high levels and had 

wide distribution. 

A wide variety of 

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The pursuit of objective (4) was confined primarily to studies of the 

disulfide reductases responsible for maintaining coenzyme A in a reduced state 

in Gram positive bacteria and for reducing 1-glutamylcysteine disulfide in 

halobacteria. At least two coenzyme A disulfide reductases have been identified 

in Bacillus cereus (Al,A5) and a 1-glutaqylcysteine disulfide reductase has 

been detected in halobacteria (P3). The latter has been purified to homogeneity - 

and is presently being characterized (A6, A. R. Sundquist-and R. C. Fahey, 

unpublished); a fuJ-1 report on this enzyme will be submitted in mid-1987. 

In conclusion, the present studies have shown that GSH metabolism arose in 

the purple bacteria and cyanobacteria where it functions to protect against 

oxygen toxicity. 

incorporated into eucaryotes via the endosymbiosis giving rise to mitochondria 

and chloroplasts. 

apparently similar functions, the thiol being coenzyme A in Gram positive 

bacteria and 1-glutamylcysteine in the halobacteria. 

prokaryotes is thus seen to be much more highly diversified than that of 

eucaryotes and much remains to be learned about this subject. 

Evidence was obtained indicating that GSH metabolism was 

Aerobic bacteria lacking GSH utilize other thiols for 

The thiol biochemistry of 

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t 

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ABSTRACTS AND PAPERS PRESENTED AT MEETINGS 

Al. Isolation and Initial Characterization of a Novel Disulfide Reductase from 

Bacillus cereus. R. C. Fahey and M. Limova, Fed. Proc. 42, 1964 (1983). 

A2. Low Molecular Weight Thiols in Halobacteria. G. L. Newton, B. J. Javor, 

and R. C. Fahey, West Coast Bacterial Physiologists Annual Asilomar 

Conference, December 15-17, 1984. 

A3. Determination of the Thiol Components of Biologically Important Thioesters 

and Disulfides. S. S. Fenton and R. C. Fahey, Fed. Proc. 44, 1211 (1985). 

Ah. Fluorescent Labeling of E. coli Thioredoxin by Monobromobimane. P. C. 

Chinn, V. Pigiet, and R. C. Fahey, Fed. Proc. 44, 1620 (1985). 

A5. Partial Purification and Characterization of an NAD( P)H Dependent Coenzyme 

A Disulfide Reductase from Bacillus cereus. T. D. Chicken and R. C. Fahey, 

West Coast Bacterial Physiolgists Annual Asilomar Conference, December 

13-15, 1985. 

A6. Partial Purification and Characterization of a Bis-1-Glutaqylcystine 

Reductase from Halobacterium halobium. A. R. Sundquist and R. C. Fahey, 

West Coast Bacterial Physiolgists Annual Asilomar Conference, December 

13-15, 1985. 

A7. Evolution of Thiol Protective Systems in Prokaryotes. R. C. Fahey and G. 

L. 

Evolution of Life, Moffett Field, California, July 23-26, 1985. 

Newton, Second Symposium on Chemical Evolution and the Origin and 

A8. Evolution of Glutathione Metabolism in Phototrophic Microorganisms. R. C. 

Fahey, R. M. Buschbacher, and G. L. Newton, Fifth ISSOL Meeting and Eighth 

International Conference on the Origin of Life, Berekely, July 21-25, 1986. 

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PUBLICATIONS 

P1. Occurrence of Low Molecular Weight Thiols in Biological Systems. R. C. 

Fahey and G. L. Newton, in "Functions of Glutathione - Biochemical, 

Physiological, and Toxicological Aspects", A. Larsson, et al, eds. , Raven 

Press, New York, pp. 252-260 (1983). 

P2. Entamoeba histolytica: A Eukaryote Without Glutathione Metabolism. R. C. 

Fahey, G. L. Newton, B. Arrick, T. Overdank-Bogart, S. B. Aley, Science, 

224, 70-72 (1984). 

P3. 1-Glutamylcysteine and Thiosulfate Are the Major Low-Molecular-Weight 

Thiols in Halobacteria. G. L. Newton and B. Javor, J. Bacteriol. 161, 

438-441 (1985 1. 

P4. Analysis of Biological Thiols: Determination of Thiol Components of 

Disulfides and Thioesters. S. S. Fenton and R. C. Fahey, Anal. Biochem. 

154, 34-42 (1986). 

P5. Determination of Thiol Proteins Using Monobromobimane Labeling and 

High-Performance Liquid Chromatographic Analysis: 

Escherichia coli Thioredoxin. P. C. Chinn, V. Pigiet, and R. C. Fahey, 

Anal. Biochem. 159, 143-149 (1986). 

Application to 

~6. The Evolution of Glutathione Metabolism in Phototrophic Microorganisms. R. 

C. Fahey, R. M. Buschbacher, and G. L. Newton, J. Mol. Evol., in press 

( 1987 

P7. Determination of Low Molecular Weight Thiols Using Monobromobimane 

Fluorescent Labeling and High-Performance Liquid Chromatography. R. C. 

Fahey and G. L. Newton, Methods Enzymol. , in press (1987). 

P8. Purification of Thiols from Biological Samples. G. L. Newton and R. C. 

Fahey, Methods Enzymol. , in press (1987). 

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