The Cytochrome Subunit Is Necessary for Covalent FAD Attachment to the Flavoprotein Subunit of p-Cresol Methylhydroxylase

Abstract

No completely satisfying explanation has been provided for the first question. The attachment of an aminoacyl group to the 8␣-or 6-position of riboflavin does not confer any unusual properties on the flavin, either free in solution or in an enzyme, with the exception of the ultraviolet-visible spectrum of 6-Scysteinylriboflavin, which is quite different from that of other forms of free and bound aminoacyl flavins (2, 3). While the oxidation-reduction potentials (E m 7 ) are about 50 -60 mV more positive for aminoacyl flavins than for unmodified forms (2), this increase in potential can also be achieved by noncovalent interactions with protein. Additionally, enzymes with covalently bound flavins do not catalyze a unique or specific set of reactions.As to the second question, it is known that 2-electron reduction of protein-free 8␣-O-tyrosylriboflavin or 8␣-S-cysteinylsulfonylriboflavin cause expulsion of the aminoacyl groups, thus producing unmodified, oxidized riboflavin (2, 4). The principle of microscopic reversibility suggests that the reverse reaction could occur within an enzyme, with or without intervention of an external enzyme. A mechanism for covalent flavinylation, which has long been in the literature, is shown in Fig. 2 (5, 6). An analogous mechanism was proposed for nonenzymic basecatalyzed nucleophilic attack at the 8␣-carbon of riboflavin derivatives in organic solvents (7).It has been suggested that covalent tethering might require prior activation of the flavin or proteins, e.g. a high energy phosphate bond (8). Of several enzymes studied to date, no specific enzyme has been implicated in the covalent modification process, which contrasts with examples of nonflavin-cofactor covalent attachment to apoenzymes (9 -13).The structural genes of several bacterial enzymes containing covalently bound flavin have been cloned into vectors for expression in new hosts: succinate dehydrogenase, fumarate reductase (14, 15), sarcosine oxidase (16), 6-hydroxy-D-nicotine oxidase (6-HDNO) 1 from Arthrobacter aerogenes (15) (all containing 8␣-N 3 -histidyl-FAD), trimethylamine dehydrogenase

Deflavination of flavo-oxidases by nucleophilic reagents

“…Similar charge^transfer complexes between FAD and Tyr residues leading to a very short £uo-rescence lifetime pattern have been shown to exist in other £avoproteins as well [25,26]. Quite interestingly, FAD could be even covalently bound to a tyrosine residue as found in the £avocytochrome subunit of p-cresol methylhydroxylase [27].…”

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

“…In either case, the heme is found to be covalently associated with the cytochrome subunit. In contrast, expression of the flavoprotein subunit alone leads to essentially deflavo enzyme even when expressed in the presence of excess FAD (Kim et al, 1995). However, when the flavoprotein and heme-containing subunits are co-expressed, or, alternatively, if cell extracts containing separately expressed subunits are mixed, flavin is found covalently associated with the flavoprotein subunit.…”

“…Recently, it was recognized that Mur B (UDP-N-acetylenolpyruvylglucosamine reductase), an enzyme required for bacterial cell wall biosynthesis (Bensen et al, 1995), has an FAD-binding fold very similar to that of PCMH, but Mur B contains noncovalently bound FAD. Several Covalent attachment of flavins to enzymes 13 other flavoproteins have moderate homology to the ADP-binding domain sequence of PCMH (Murzin, 1996).A role for the function of the Sa-0-tyrosyl FAD of PCMH has been proposed recently (Kim et al, 1995). Inspection of the crystallographic structure of PCMH reveals that the covalent link is located within a probable electron transfer pathway between the flavin and the c-type cytochrome (Fig.…”

YgfX (CptA) is a multimeric membrane protein that interacts with the succinate dehydrogenase assembly factor SdhE (YgfY)

“…The loss of Sdh5/SdhE prevents FAD incorporation into Sdh1/SdhA and results in a nonfunctional SDH (Hao et al, 2009;McNeil et al, 2012). The process of flavinylation has traditionally been thought of as autocatalytic (Edmondson & Newton-Vinson, 2001;Heuts et al, 2009;Kim et al, 1995;Mewies et al, 1998). Consequently, SdhE was the first FAD assembly factor identified in bacteria (McNeil et al, 2012).…”