A Cysteine Cluster Critical for Flavin Binding in Flavocytochrome b2 from Baker's Yeast

Abstract

We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome bz, a reaction which leads to the loss of flavin-binding capacity ('inactivation') [D. Pompon and F. Lederer (1982) Eur. J . Bioclienz. 12Y,1371. For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit. In this work we report the results of sequence studies which elucidate the nature of the modification. The modified protein was cleaved with cyanogen bromide and the peptidcs separated on Sephadex G-I 00 and SP-Sephadex C-25. ''C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting.It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion. Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule. Only two of the possible cross-links were found. These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme. These results, combined with those obtained in the affinity labeling reaction of holoenzyine with brornopyruvate [Alliel et al. (1982) Eur. J. Biochrm. 122,553 -5581, show that the three residues are located in or close to the active site. Their possible role is discussed.We recently showed that flavin-free flavocytochrome hZ reacts rapidly with 2-keto-3-butynoate to form a covalent adduct which is n o longer active in the presence of added F M N [l]. This loss of activity appears to result essentially from the loss of flavin binding capacity. The cofactor itself affords protection against inactivation and the native enzyme is not affected under the same conditions. The inactivated enzyme was found to have incorporated about one mol reagent/mol subunit, most probably on a cysteine residue.We report in this paper results of peptide isolation experiments. They establish that indeed the modification affects cysteine, but that three different residues in the sequence are. fractionally labeled. Furthermore we provide evidence that these cysteines are close together in space in the flavin-free en.zyme and hence probably also in the native enzyme. MATERIALS A N D METHODS M a trrialsThe preparation of flavocytochrome hZ and its flavin-free derivative were described in our previous paper Analytical Metliod.~Peptide and protein hydrolyses were carried out at 110 "C in 5.7 M HCI, 0.1 phenol, in evacuated sealed tubes. Amino acid compositions were determined with an LKB 4400 amino acid analyser, operated at absorbance = 0.1 for full scale with a normal ninhydrin system. The recorder of the analyser was used to calculate proline, and the 570-nm channel was recorded on a Delsi 510 recorder coupled with a Delsi ICAP 10 integrator. The amounts analysed ranged over 0.2-1.5 nmol. At that high sensitivity, values for serine, glutamic acid and glycine...

Primary structure of flavocytochrome b2 from baker's yeast

“…In that work, aCB3 residues 10 and 11 were identified as Gln instead of Glu. Since two automated degradations of two different aCB3 preparations led us to identify Glu at those positions [70], we believe this is the correct identification.…”

“…Affinity labeling with bromopyruvate led to the conclusion that cysteine-233 was located in the active site and that its modification resulted in enzyme inactivation [23]. Modification of flavin-free enzyme with 2-keto-3-butynoate led to mutually exclusive labeling of cysteines 200,216 and 233, as well as to cross-linking between cysteines 200 and 216 and cysteines 200 and 233 [69,70]. The interpretation was that the modification of cysteines 200 and 216 led to the loss of flavin-binding capacity and furthermore that the three cysteines were close to each other in space (even though the idea that cross-linked cysteines 216 and 233 belong to different subunits cannot be rigorously excluded); therefore they were close to the active site if not in it.…”

Complete amino acid sequence of flavocytochrome b2 from baker's yeast

“…3). So far, all the residues which were specifically labeled with bromopyruvate [30], with ketobutynoate [32] and with phenylglyoxal [42], and found to play a role at the active site and in flavin binding, are located in with an averaging window of seven residues. The potential for forming a helix, {i sheet and /i turns was calculated according to [40].…”

“…Samples of CNBr blocked with il-~4C1-2-keto-3-butynoate (31,32) were kindly given by D r D. Pompon; they were used for some of the experiments.…”

Nondestructive laser vaporization of high molecular weight, single-stranded DNA

“…The distribution is peaked in the normal direction and is considerably tighter than a simple cosine distribution expected for a thermal desorption process. 13 Third, the 32P present on the filter paper is evenly distributed as expected for molecular vaporization. Spallation, or the removal of macroscopic pieces of the mixture, has been shown in prior studies6 to lead to a spotted or speckled appearance.14 Taken together, the images of the distribution obtained in these experiments strongly suggests that individual molecules are being vaporized.…”