Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors
Introduction
The structure of proteins in the crystalline state is usually recognized as a faithful model of their conformation in the physiological milieu and is therefore exploited to gain a better understanding of function in molecular terms. This view assumes that the precipitating agents present in the mother liquor and the intermolecular interactions that stabilize the crystal lattice do not select an inactive conformer among those coexisting in solution. A useful criterion to verify this assumption is retention of function in the crystal [1].
A technique that permits measurements of function-related parameters on the same single crystals that are used for X-ray crystallography, when the protein contains a chromophoric reporter group, such as a coenzyme or heme, is polarized absorption microspectrophotometry (Ref. [1] and references therein). In the case of redox enzymes and electron carriers, the characteristic absorption spectra of redox centers allow the monitoring of catalysis as well as intra- and intermolecular electron transfer in multicenter proteins and multiprotein complexes.
While many individual redox enzymes and electron transport proteins have been crystallized and their structures determined to high resolution, only a few multiprotein complexes have been similarly characterized, among which the binary complex between Paracoccus denitrificans methylamine dehydrogenase (MADH) and its electron acceptor, the blue copper protein amicyanin [2], and the ternary complex between the same two proteins and cytochrome c-551i [3], [4]. Their three-dimensional structures and those of a few other natural and model systems provide a basis to attempt the description of pathways for intra- and intermolecular electron transfer.
MADH [5] is a tetrameric protein composed of two heavy and two light subunits. Each light subunit contains tryptophan–tryptophylquinone (TTQ), a cofactor that results from the covalent linkage of two tryptophan side chains, one of which has been modified to contain an orthoquinone function [6]. The oxidation of methylamine catalyzed by MADH produces formaldehyde and the two-electron reduced (aminoquinol) form of TTQ. The coenzyme is reoxidized by the one-electron carrier amicyanin in two subsequent steps, the first one leading to formation of the aminosemiquinone form of TTQ. Reduced amicyanin is, in turn, oxidized by a soluble cytochrome that carries the electron to a membrane-bound oxidase.
The catalytic mechanism of MADH and the rates of electron transfer, in solution, between either the fully reduced or the semiquinone forms of TTQ and amicyanin, as well as between the reduced binary complex and cytochrome c-551i, have been investigated in detail. The observed electron transfer rate constants, in the order of 102 s−1, have been analyzed and discussed in light of electron transfer theories ([7], [8] and references therein) and of the structures determined in the crystalline state.
Both the binary and the ternary complex of MADH with its electron acceptors have been examined by polarized single crystal absorption microspectrophotometry [9], [10]. The data show that TTQ is reduced by methylamine and, therefore, that crystalline MADH retains catalytic activity. Furthermore, the spectral changes of TTQ, copper and heme indicate that intermolecular electron transfers from reduced TTQ to copper and from the latter to heme are permitted, under appropriate experimental conditions.
These results may suggest, but do not prove, that specific intermolecular recognition is not drastically altered by the crystallization medium and that the observed contacts between redox partners correspond to those that support coordinated function in solution. Rate measurements in the crystal would provide a more stringent criterion to compare function and, hence, structure in the two physical states.
If the rates of electron transfer within the crystalline binary and ternary complexes were as high as in solution, where the encounter and transient association of redox partners mediate the process, kinetic measurements would be inaccessible to microspectrophotometry, under our experimental conditions. However, the distance of approximately 24 Å between the copper and the heme iron, within the rigid geometric arrangement of the ternary complex in the crystal, is usually regarded as too long for a biologically efficient electron transfer. It was therefore of interest to explore whether the rate of heme reduction in the crystal might be lower than in solution.
In the present communication, together with the results of a kinetic study of heme reduction by polarized single crystal absorption microspectrophotometry, we report the preliminary results of a related study by continuous wave electron paramagnetic resonance (EPR) on polycrystalline powders of the binary and ternary complexes. The EPR experiments were designed mainly to detect the redox states of TTQ and copper in the ternary complex in which their relevant optical signals escape direct observation because of the presence of the prevailing heme absorption.
Section snippets
Materials and methods
Proteins were provided by Professor V.L. Davidson, University of Mississippi Medical Center, Jackson, MS. Crystals were provided by Professor F.S. Mathews, Washington University Medical School, St. Louis, MO, or obtained according to published procedures [2], [3], [4]. All crystals used in the present experiments grew in a mother liquor containing approximately 2.3 M phosphate, at pH 5.7. In view of manipulation for the microspectrophotometric and EPR measurements, they were stabilized in 3 M
Results
Previous microspectrophotometric studies [9] on crystals of the binary complex between MADH and copper-depleted amicyanin had shown that the enzyme is catalytically active toward methylamine and that all TTQ centers undergo a two-electron reduction to the aminoquinol form. From the intensities of TTQ polarized absorptions along the principal optical directions of the crystal, it is possible to determine the orientations of the various transition moments with respect to the crystal axes. The
Discussion
The aim of the present work was to inquire whether the three-dimensional structures and the intermolecular contacts present in the crystals of the binary and ternary complexes between MADH and its electron acceptors provide a realistic basis to identify the physiological pathways of electron transfer from TTQ to copper and from copper to the heme iron. The criterion chosen to answer the question was retention of function in the crystal. Kinetic parameters allow for a quantitative comparison of
Acknowledgements
The Authors are grateful to Professors V.L. Davidson and F.S. Mathews for providing proteins and crystals prepared in their laboratories and for valuable discussions.
Research funded by MIUR and the National Research Council of Italy.
References (21)
- et al.
Enzymatic and electron transfer activities in crystalline protein complexes
J. Biol. Chem.
(1996) - et al.
Electron transfers in chemistry and biology
Biochim. Biophys. Acta
(1985) - et al.
Protein dynamics enhance electronic coupling in electron transfer complexes
J. Biol. Chem.
(2001) - et al.
Protein function in the crystal
Annu. Rev. Biophys. Biomol. Struct.
(1996) - et al.
Crystal structure of an electron-transfer complex between methylamine dehydrogenase and amicyanin
Biochemistry
(1992) - et al.
Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein and a c-type cytochrome
Protein Sci.
(1993) - et al.
Structure of an electron transfer complex: methylamine dehydrogenase, amicyanin, and cytochrome c551i
Science
(1994) Methylamine dehydrogenase
- et al.
A new cofactor in a prokaryotic enzyme: tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase
Science
(1991) Methylamine dehydrogenase: structure and function of electron transfer complexes
Sub-cell. Biochem.
(2000)
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