Biomimetic Chemistry - American Chemical Society

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7

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch007

Flavin Coenzyme Analogs as Probes of Flavoenzyme Reaction Mechanisms C. WALSH, F. JACOBSON, and C. C. RYERSON Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

The use of riboflavin coenzyme analogs as biomimetic probes offlavoenzyme reaction mechanisms has yielded new information about these biological redox processes. Substitution of carbon at the 1 or 5 positions for the nitrogens normally present in the flavin isoalloxazine ring system leads to dramatic alterations in redox properties but does not affect strongly recognition and binding by a variety of apoflavoproteins. 5-Carba-5deazaflavins are catalytically competent coenzymes only with flavoenzymes that catalyze redox conversions involving two-electron transfer steps. Thus some dehydrogenases but no flavoprotein oxidases or monooxygenases are functional for turnover. In contrast, 1-carba-1-deazaflavin analogs, with accessible one-electron chemistry, function catalytically in all classes of flavoenzymes examined. The 1-deaza system is more difficult to reduce, so lower V 's with some enzymes may reveal that the redox steps are rate determining. When monooxygenases are reconstituted with 1-deazaFAD, some still show NADH oxidation coupled to specific substrate hydroxylation. Studies with synthetic 8hydroxy-7-demethyl-5-deazariboflavin are presented to confirm the identity of this 5-deazaflavin chromophore in the novel redox coenzyme, Factor 420, from methanogenic bacteria. max

T

he central topic of this volume is biomimetic chemistry. In this chapter we describe our recent studies using synthetic analogs of riboflavin-based coenzymes with specific atom substitutions in the 0-8412-0514-0/80/33-191-119$05.00/0 © 1980 American Chemical Society

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

120

BIOMIMETIC CHEMISTRY

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch007

tricyclic isoalloxazine ring system to dissect and analyze the chemical features that permit the utilization of flavin coenzymes in a wide range of biological oxidation-reduction processes. An initial summary of the nature and scope of redox processes involving flavin coenzymes will be presented to set the framework for evaluation of the utility of the specific coenzyme analogs. Analogs altered atN(5), atN(l), or atN(l) andN(5) by synthetic substitution of carbon will be discussed before analysis of 8-demethyl-8-hydroxy analogs either with nitrogen or carbon at the 5 position. Roles of Flavin Cofactors in Redox Enzymology The functional end of the flavin coenzymes F M N and F A D is the tricyclic isoalloxazine system, with the numbering system shown in structure I, the air-stable, yellow, oxidized form. The other two functionally important redox states are the one-electron-reduced semiquinone, II ( p K = 8.4 for dissociation at N(5)), and the twoelectron-reduced, colorless dihydroflavin, III. In the dihydro form N(5), C(4a), C(la), andN(l) form a diaminoethylene system and it was anticipated that nitrogen at the 5 and 1 positions would be key to coenzymatic function. a

The ability to form a stable one-electron-reduced radical (semiquinone) allows flavin cofactors to sit at the crossroads of two-electron and one-electron transfer chains. That is, they can be reduced by organic substrates two electrons at a time and be reoxidized by either obligate one-electron acceptors such as cytochromes (e.g., yeast cytochrome b or cytochrome b reductase/cytochrome b ) and ironsulfur cluster proteins (adrenodoxin reductase/adrenodoxin) or by facultative one-electron acceptors such as benzoquinones (coenzyme 2

5

5

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch007

7.

WALSH E T A L .

121

Flavin Coenzyme Analogs

Q) and naphthoquinones (vitamin K) (1,2). These enzymes, in keeping with the membraneous nature of the reoxidants (small molecules or proteins), are often membrane-associated and are classified as dehydrogenases (e.g., succinate dehydrogenase). Other flavoenzymes are soluble and can be reoxidized by apparent two-electron acceptors such as N A D (e.g., transhydrogenase). A second major category of flavoenzymes (soluble) are those where the enzyme-bound dihydroflavin is reoxidized by molecular oxygen (1, 2, 3). Net two-electron transfer yields I and hydrogen peroxide, H 0 . These are the flavoprotein oxidases (e.g., D- and L-amino-acid oxidases, amine oxidase, glucose oxidase). A variant of the flavoenzymes reacting with 0 are certain bacterial enzymes that carry out monooxygenation, transfer of one oxygen atom to product. These flavoenzyme monooxygenases thus split 0 and show activity towards two kinds of substrates: activated aromatic rings (phenol, p-hydroxybenzoate, salicylate) and ketones (cyclohexanone). The sole mammalian flavoprotein monooxygenase reported is a microsomal N-oxidase, converting, for example, tertiary amines to amine-N-oxides (4). Typical oxidase and monooxygenase reactions are shown in Table I. The mechanisms for passage of electrons out of the cosubstrate undergoing oxidation and into the flavin undergoing reduction have been scrutinized carefully in the past decade, as have those for reoxidation of flavin and passage of electrons to the cosubstrate that experiences net reduction. Depending on substrate structure a variety of covalent intermediates have been postulated or demonstrated directly: thus 0 reacts with dihydroflavins to form 4a flavin hydroperoxides IV 2

2

2

2

2

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

2

2

z

Substrates

2

CH —NH + O

2

<

2

/

Q -N

+ 0

X +

/

O

2 +

NADPH

4- NADPH

2

H O — \ — C O O " + 0 + NADPH

H

I

R—C—COO" + 0

NH

Substrates

2

z

2

+

2

2

NADP+ + H 0 +

NADP+ + H 0 + 2

2

O

o

3

S + 2e" + 2 H ) NADH/flavin oxidoreductase N-methylglutamate synthetase

Enzyme

Table III.

535,365

400,340

445,375

3

1

Yes 1 and 2 e" 3.9 sec" -311 mV No (None atC(l))

No 2 e" only