Isolation and characterization of methanophenazine and function of phenazines in membrane-bound electron transport of Methanosarcina mazei Gö1

Abstract

A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F42OH2 dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F420H2 was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. The Km values for 2-hydroxyphenazine and phenazine were 35 and 250 microM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F420-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1.

FIG. 1

HPLC of isooctane extract prepared from lyophilized membranes of M. mazei Gö1. For separation conditions, see Materials and Methods.

FIG. 2

UV-Vis spectrum of methanophenazine in the oxidized (curve 1) and reduced (curve 2) forms. For assay conditions, see Materials and Methods.

FIG. 3

¹H NMR spectrum of methanophenazine from M. mazei Gö1 (CD₃OD; 499.9 MHz). (Inset) Assignment of the ¹H NMR signals.

FIG. 4

¹³C APT NMR spectrum of methanophenazine from M. mazei Gö1 (C₆D₆; 125.71 MHz). (Inset) Assignment of the ¹³C NMR signals.

FIG. 5

Coupling of 2-hydroxyphenazine reduction by H₂ (A) and its oxidation by heterodisulfide under N₂ (B) as catalyzed by washed membranes from M. mazei Gö1. Washed membranes (12 μg of protein) were suspended in 1 ml of 25 mM MOPS buffer (pH 7) containing 2 mM dithioerythritol and 1 mg of resazurin per liter under a hydrogen atmosphere. The reaction was started by the addition of 6 μl of a 2-hydroxyphenazine solution (20 mM in ethanol-acetic acid [1:1, vol/vol]). After 15 min, the cuvette was flushed with nitrogen for 30 min and 2 μl of CoB-S-S-CoM (90 mM) was added. The reaction was monitored at 425 nm as described in Materials and Methods.

FIG. 6

Tentative scheme of membrane-bound electron transport in M. mazei Gö1. Cytb₁ and Cytb₂, cytochromes b₁ and b₂, respectively.