Publisher Summary This chapter discusses the fluorescence and optical characteristics of reduced flavins and flavoproteins. Flavins and flavoproteins in their oxidized state (Flex) are typically yellow, with the absorption spectrum in the visible and near-ultraviolet (UV) range being characterized, by two well-resolved bands centered around 450 and 370 nm. This chromophore, free in solution, exhibits a strong fluorescence that is maximal around 520 nm; when the chromophore is bound to flavoproteins, its emission can be fully quenched. Studies show that the near-UV absorption spectrum of the 4a,5-dihydroflavin chromophore is similar, in its appearance, to that of its 1,5-dihydro isomer, but its shape is even more dependent on temperature and solvent. In particular, substituents that have an effect on the internal mobility of the molecule were found to cause major spectral changes on the chromophore free in solution and also when bound to various apoflavoproteins. At ambient temperature, 4a,5-dihydroflavins show a weak fluorescence only when an appropriate substituent decreases their internal mobility. However, at low temperature in rigid ethanol glasses or when bound to apoflavoproteins, 4a,5-dihydroflavins exhibit a rather strong fluorescence emission maximal in the range 480-530 nm.

/pdf/fluorescence-and-optical-characteristics-of-reduced-flavines-3bgb6j4zr8.pdf

Fluorescence and optical characteristics of reduced flavines and flavoproteins.

The last decade has produced a veritable explosion of knowledge on the chemical reactivity of the isoalloxazine ring system of the flavin coenzymes, largely because of a side-by-side development with knowledge of flavoenzymes and model flavin studies. In this period the visible absorption spectra and epr spectra of the flavin semiquinone in its cationic, neutral, and anionic forms have been defined'-" (FIGURE 1 ) . In the same period a large number of flavoenzymes have been investigated by rapid reaction spectrophotometric techniques, and in nearly all cases distinctive spectra characterized by longwavelength-absorption bands have been detected as catalytic intermediates. With few exceptions, the spectra of the intermediates are unequivocally different from those of flavin semiquinone in either its neutral or its anionic form. As will be discussed in more detail below, these intermediates are in general readily thought to contain the flavin in the oxidized or reduced state, on the basis of their spectral properties. During this period the great chemical reactivity of the flavin system has been uncovered.',8 This model work has led to the synthesis of well-defined substituted isoalloxazines, with substituents at almost every atom of the isoalloxazine ring system. The recognition of this great reactivity has led to the obvious suggestion that intermediates in flavoenzyme catalysis involve covalent linkage of substrate moieties with the flavin.'-'l While the formation of such intermediates cannot be excluded rigorously on the basis of present evidence, the known spectral properties of such model substituted flavins fail to account for the intermediates observed in flavoenzyme catalysis.

/pdf/role-of-charge-transfer-interactions-in-flavoprotein-48k9wic1zu.pdf

Role of charge‐transfer interactions in flavoprotein catalysis

Publisher Summary This chapter discusses two classes of flavoproteins that function solely to mediate electron transfer between the prosthetic groups of other proteins. According to the function the protein was termed electron-transferring flavoprotein. More recently, a different class of flavoprotein carriers has been isolated from microorganisms.These proteins have been called “flavodoxins” because of their functional interchangeability with the ferredoxins. The chapter describes the flavodoxins because they are the first flavoproteins for which three-dimensional structures have been determined. Organisms from which flavoproteins of the flavodoxin type have been isolated include several strictly anerobic bacteria, representatives from the obligately aerobic, facultatively anaerobic, and photosynthetic. Flavodoxins do not react directly with small molecules, such as the pyridine nucleotides, and their only known biochemical substrates are other redox proteins. The relative activities of flavodoxins and ferredoxins as electron carriers have been determined in both plant and microbial systems with results, which indicate that transfer rates vary somewhat according to the source of the carrier.

2 Flavodoxins and Electron-Transferring Flavoproteins

In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5 Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given

Preparation, characterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphate, and 5-deazariboflavin 5'-diphosphate, 5'leads to5'-adenosine ester.

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X-ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816). This form of TrxR, denoted FO, permits the reaction of enzyme-bound reduced FAD with a redox-active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X-ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34 degree "butterfly" bend about the N(5)-N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15-28 degrees for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side-chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.

https://onlinelibrary.wiley.com/doi/pdf/10.1110/ps.8.11.2366

Crystal structure of reduced thioredoxin reductase from Escherichia coli: structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor.

The pyramidal inversion of the N(5)-centre of several reduced flavins was measured by NMR. The inversion barrier was found to be ∼10 kcal/mol in acetone solutions and to be independent of the size of the N(5) substituent. An increase of the inversion barrier of ∼5 kcal/mol was observed in the case where the N(5) substituent could only be in axial position, and an increase of ∼3.5 kcal/mol was observed for an acyl-like N(5) substituent. In aqueous solution the inversion barrier increases by ∼3 kcal/mol. The stereochemistry of reduced flavin and its potential relevance in flavin-dependent biological dehydrogenations is discussed.

/pdf/nmr-study-of-nitrogen-inversion-and-conformation-of-1-5-lass12q9c6.pdf

Ludwig Tauscher

Papers

NMR.‐Study of Nitrogen Inversion and Conformation of 1,5‐Dihydro‐isoalloxazines (‘Reduced Flavin’). Studies in the flavin series, XIX. Communication

Flavin-reihen 19. mitt. nmr-untersuchung der stickstoffinversion und konform. von 1,5-dihydro-isoalloxazinen (′reduziertes flavin′)