Characterization of the tryptophan-derived quinone cofactor of methylamine dehydrogenase by resonance Raman spectroscopy

Abstract

The resonance Raman (RR) spectrum of oxidized methylamine dehydrogenase (MADHOX) exhibits a set of C-H, C-C, C = C, and C = O vibrational modes between 900 and 1700 cm-1 that are characteristic of the quinone moiety of the tryptophan tryptophlyquinone (TTQ) cofactor. The close similarity of the RR spectra for MADHs from Paracoccus denitrificans (Pd), Thiobacillus versutus (Tv), and bacterium W3A1 proves that the same cofactor is present in all three proteins. The MADHs from Pd and Tv have a v(C = O) mode at approximately 1625 cm-1 that shifts approximately 20 cm-1 upon 18O substitution of one of the carbonyl oxygens and is assigned to the in-phase symmetric stretch of the two C = O groups. The semiquinone form of Pd MADH has its own characteristic RR spectrum with altered peak frequencies and intensities as well as a decrease in the total number of peaks. The hydroxide and ammonia adducts of MADHOX produce RR spectra similar to that of the semiquinone. The spectral changes in all three cases are interpreted as being due to reduced conjugation of the cofactor. The ammonia adduct is formulated as a carbinolamine, a likely intermediate in the enzymatic mechanism. In contrast, formation of the electron-transfer complex between amicyanin and MADHOX has no effect on the vibrational frequencies (and, hence, structure) of either the MADH quinone or the amicyanin blue copper site. The behavior of the TTQ cofactors of Pd and Tv MADHs are very similar to one another and somewhat different from W3A1 MADH, particularly with regard to adduct formation and ability to undergo isotope exchange with solvent. These differences are ascribed to the cofactor environments within the proteins rather than to the structure of the cofactor itself.

Quinoproteins: enzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or tryptophan‐tryptophan quinone

“…This confirms the identity of the cofactors and suggests similar environments, in line with the high amino acid sequence similarity of the small subunits [26,27]. Although the absorption spectra of all redox forms of the enzymes are also very similar, differences are observed when ammonium salts are added to the oxidized form and in isotope-exchange experiments [115,116]. This might indicate small differences in local environments due to, for example, the close vicinity of an amino acid residue in certain cases.…”

“…Also based on these structural data, the carbonyl group at the C(6) position is most accessible to solvent so that it might have a similar role to the C(5)-carbonyl group in PQQ. Resonance Raman spectroscopy of enzyme treated with ammonia suggests that, depending on the source of the enzyme, either the iminoquinone or carbinolainine adduct of TTQ are formed [115,1161. From this it is conceivable that the aminophenol form of TTQHz and TTQH' also exist.…”

Protein Radicals in Enzyme Catalysis

“…24,513 The amine is proposed to form a Schiff base at C6 of TTQ, detected as a transient intermediate at 425 nm when the reaction is run in the absence of amicyanin. 514 As in the case of BPAO, 515 a large kinetic isotope effect (k H / k D ) 17.2) 516 was detected on removal of a proton by a general base catalyst thought to be Asp70 (T. versutus), again in analogy with AOs.…”

Computational Approaches to Understanding the Self‐assembly of Peptide‐based Nanostructures

“…[26] This approach is particularly useful in confirming the validity of proposed structures based on experimental data that also provides structural insight, such as IR, UV-Vis absorption, circular dichroism, etc. However, it is also possible for AAFF MD studies to generate potential structures by carrying out longer timescale (100 ns Ð 1 µs) simulations on smaller systems (typically 10Õs of monomers) to gain an insight into what the dominant interactions between the sub-units and the stability of the resulting aggregates.…”