Yeast lactate dehydrogenase, cytochrome

Many type b cytochromes associated with the classic cytochrome oxidase (a and 03) show their a peaks at 561-563 nm, and they are usually designated as cytochrome b. The name cytochrome 61 was originally used for the pigments with a peak at 557-560 nm, but it is now generally applied to the cytochrome in the nitrate reductase system. Cytochrome 62 (a, 557 nm) is the entity of yeast lactate dehydrogenase which contains FMN and protoheme. Cytochromes 63 (a, 559 nm) and 65 (a, 556 nm) participate in the microsomal electron transport system in plants and animals, respectively. Both the primary and ternary structures of cytochrome 65 are known. Cytochromes bg (a, 563 nm) and 6-559 are the components of the photosynthetic electron transport system in plant. 

Cytochrome 62 is also called yeast lactate dehydrogenase (EC 1.1.2.3, lactate cytochrome c oxidoreductase) (204 205]. This cytochrome possesses both FMN and protoheme (206-211). Although the native enzyme also contains deoxyribonucleic acid, this moiety can be removed by deoxyribonuclease without affecting the enzymic activity of the enzyme (212) and is supposed to be a heterogeneous component (213). This enzyme completely differs in its heme content from animal lactate dehydrogenase and also from yeast n(—)-lactate dehydrogenase. 

In contrast to the flavin oxidases, flavin dehydrogenases pass electrons to carriers within electron transport chains and the flavin does not react with 02. Examples include a bacterial trimethylamine dehydrogenase (Fig. 15-9) which contains an iron-sulfur duster that serves as the immediate electron acceptor167 169 and yeast flavocytochrome b2, a lactate dehydrogenase that passes electrons to a built-in heme group which can then pass the electrons to an external acceptor, another heme in cytochrome c.170-173 Like glycolate oxidase, these enzymes bind their flavin coenzyme at the ends of 8-stranded a(i barrels similar... 

Baker s yeast contains at least four lactate dehydrogenases, three of which are located in the mitochondrion and one in the cytoplasm (Table 1). Electron flow in the mitochondrial enzymes is linked to cytochromes rather than nicotinamides. Their apparent physiological function is the reversible interconversion of pyruvate and lactate, and it is not clear whether any of these four enzymes accept ketones other than pyruvate, which would limit their importance in organic synthesis. 

Three types of lactate dehydrogenase are found in yeast, which may be considered as metal-containing flavoproteins. These are L-lactate cytochrome c reductase or cytochrome b, D-lactate dehydrogenase, which is found in anaerobic yeast, and D-lactate cytochrome c reductase, which is associated with the mitochondria of aerobic cells. 

This enzyme [also known at l(-[-)-lactate dehydrogenase] was first extracted from bakers yeast by Bernheim in 1928 (272). Bach et al. (273) showed in 1942 that lactate dehydrogenase copurified with a species of cytochrome b, which contained protoheme as prosthetic group. The... 

Cytochrome 62 is stereospecific for l(- -)-lactate. It also oxidizes other a-hydroxymonocarboxylic acids at slow rates 80, 96). As electron acceptors ferricyanide, methylene blue, 2,6-dichloroindophenol, 1,2-naphthoquinone 4-sulfonate, and cytochrome c have been used. This wide acceptor specificity is characteristic of a number of flavoproteins, which are generally capable of reducing quinoid structures and ferric compounds 97). However, as will be seen below, cytochrome c is considered to be the physiological electron acceptor for the yeast L-lactate dehydrogenase. 

The role of L-lactate dehydrogenase in the physiology of aerobic yeast is not clear. It has been shown that its presence in yeast depends on the availability of oxygen (306), and that in the presence of antimycin A, which inhibits electron transfer to cytochrome c from NADH-linked substrates, L-or D-lactate can partially support the growth of Saccharo-myces cerevisiae (307). Under these conditions, cyanide inhibited the growth. Therefore, it has been concluded that l- and D-lactate-cyto-chrome c reductases can feed electrons to the respiratory chain at the level of cytochrome c and provide energy through the third site of oxidative phosphorylation (307). 

Flavocytochromes 2 2-hydroxyacid dehydrogenases found in the inter-membrane space of yeast mitochondria where they couple oxidation of the substrate to reduction of cytochrome c. Examples include the enzymes from Saccharomyces cerevisiae and Hansenula anomala, both of which are l-lactate dehydrogenases (Chapman et al., 1998), and the enzyme from Rhodotorula graminis which is a L-mandelate dehydrogenase (Ilias et al., 1998). This article will concentrate on the flavocytochrome 2 (L-lactate cytochrome c oxidoreductase) from S. cerevisiae (Bakersi yeast), since this is by far the most studied of these enzymes (Chapman et al., 1991). Therefore, throughout this article, the term flavocytochrome 2 will refer specifically to the enzyme from S. cerevisiae unless otherwise stated. 

In 1915, Harden and Norris observed that dried yeast, when mixed with lactic acid, reduced methylene blue and formed pyruvic acid 4). Thirteen years later Bernheim prepared an extract from acetone-dried baker s yeast, which had lactate dehydrogenase activity (5). Bach and co-workers demonstrated that the lactate dehydrogenase activity was associated with a 6-type cytochrome, which they named cytochrome 62 (6). In 1954, the enzyme was crystallized, enabling the preparation of pure material and the identification of flavin mononucleotide as a second prosthetic group (2). Since then, significant advances have been made in the analysis of the structure and function of the enzyme. Much of the earlier work on flavocytochrome 62 has already been summarized in previous review articles (7-10). In this article we shall describe recent developments in the study of this enzyme, ranging fi om kinetic, spectroscopic, and structural data to the impact of recombinant DNA technology. 

It was discovered in 1958 that anaerobically grown yeast contains a form of lactate dehydrogenase which is different from the d- and L-lac-tate cytochrome c reductases of aerobic yeast (306, 319). The enzyme has been partially purified (320, 321), and shown to contain flavin (320-322). Gel filtration studies have suggested a molecular weight of about 100,000 (320, 321). Preparations of the enzyme oxidize several d-2-hydroxyacids to the respective keto acids in a reversible manner (320). For the forward reaction, ferricyanide, 2,6-dichloroindophenol, menadione, and methylene blue have been used as electron acceptors, and for the reverse reaction leucomethyl viologen and FMNHa are effective electron donors (320). A number of L-2-hydroxyacids and 2-keto acids have been shown to be competitive inhibitors. Oxalate, cyanide, o-phenanthro-line, and EDTA are also potent inhibitors (320, 321, 323, 324). The inhibition by metal chelators develops slowly and is reversed by addition of Zn, Co, Mn +, or Fe + (320, 323-326). Substrates prevent the inhibition by chelators at concentrations considerably lower than their respective Km values (327). It has been suggested that EDTA inactivation involves the removal of a metal, most probably Zn +, from the substrate binding site of the enzyme (325, 326, 328, 329). However, others have... 

Cytochrome 2 functions in yeasts both as an electron transport system and as lactate dehydrogenase, combining the functions of two different proteins—L-lactate dehydrogenase and cytochrome b—into a single catalytic unit. The molecular mass of the enzyme is approximately 235,000. It consists of four subunits, each containing a group of flavin mononucleotide (FMN) and heme. FMN can be reversibly separated from the protein. The absorption spectrum of 2 corresponds to a low-spin complex. It is assumed that the transfer of electrons from lactate proceeds through flavin to the heme. ... 

Since mitochondrial cytochrome c was available commercially (horse heart muscle being the most common source) and could readily be purified to a high level, it formed the basic subject for most of the pioneering studies. Many ideas concerning the electrochemical mechanism, in particular, the mode of interaction with the electrode, have developed around the considerable wealth of information that is available [14, 18] on the structure and properties of the protein molecule. The extent to which the metal centre is buried is illustrated well in Fig. 1 which shows the 3D structure [19] of yeast (iso-1) cytochrome c and a view of the exposed active site. The major function of cytochrome c is as electron donor to cytochrome c oxidase (Complex IV), the membrane-bound enzyme that is the terminus of the aerobic respiratory chain and a site for proton translocation. Another physiological oxidant of cytochrome c (in yeasts) is cytochrome c peroxidase, a soluble enzyme whose crystal structure is known (see Sect. 7). The most important reduc-tant of cytochrome c is the cytochrome Cj component of the membrane-bound hcj complex (Complex III), but others (see Sect. 6, Scheme 5) include cytochrome b, sulfite oxidase, and flavocytochrome (lactate dehydrogenase, found in yeasts). 

Lactic dehydrogenase in yeast differs from the muscle enzyme in the lack of a requirement for, or reaction with, DPN. The yeast enzyme has been purified by Bach, Dixon, and Zerfas and found to be associated with a hemoprotein with reduced bands at 530 and 556 m/x which has been designated as cytochrome b2. The reduced bands appear instantly on addition of lactate, but, as in the case of heart muscle succinic dehydrogenase, the identity of the hemoprotein with lactic dehydrogenase has not been fully established. Crude preparations will reduce cytochrome c, but this activity is diminished on purification. An additional factor is thought to be required for the reaction with cytochrome c. The reduced cytochrome b2 is slowly reoxidized by air. 

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