Isolation and characterization of the cytochrome domain of flavocytochrome b2 expressed independently in Escherichia coli

Abstract

The cytochrome domain of flavocytochrome b2 (L-lactate dehydrogenase) was expressed in the bacterium Escherichia coli and a purification procedure was developed. When expressed in E. coli, the b2-cytochrome domain contains protohaem IX and has an electronic absorption spectrum identical with that of the cytochrome b2 'core' produced by proteolytic cleavage of the enzyme isolated from yeast. The b2-cytochrome domain isolated from E. coli has an Mr of 10,500 and a redox potential of -31 +/- 2 mV. High-field n.m.r. studies indicate pKa values for the haem propionate groups to be 4.8 and 4.6, consistent with these groups being exposed to solvent rather than buried inside the protein. Using n.m.r. spectroscopy, we have determined an electron self-exchange rate constant for the b2-cytochrome domain of 2.3 x 10(6) M-1.s-1, which is more than two orders of magnitude larger than the value obtained for microsomal cytochrome b5, a homologue of b2-cytochrome domain.

Microbial L‐ and D‐Lactate Selective Oxidoreductases as a Very Prospective but Still Uncommon Tool in Commercial Biosensors

“…[42] The protoheme IX-containing domain of cytochrome b 2 of S. cerevisiae has a redox potential of À 31 mV, which is very close to the published value of À 28 mV determined for the proteolytically released cytochrome b 2 core. [43] A similar low mid-point potential E h = À 19 mV was reported for FC b 2 -heme from H. anomala. [44] A low redox potential of FC b 2 is a favorable feature for biocatalytic chromate reduction, which is characterized by particularly high redox potential (E 0 = + 1.3 V).…”

Purification and Characterization of an Extracellular Heme-Binding Protein, HasA, Involved in Heme Iron Acquisition

“…The absorption spectra of the fractions containing HasA showed a pattern typical of a hemoprotein with a peak at 277 nm and another one in the Soret region, at 407 nm (Figure 2, trace A). However, pure fractions presented abnormally high A 277 /A 407 ratios, ranging from 2.1 to 2.5, instead of less than 1, the value usually observed for hemoproteins (Di Iorio, 1981;Lee et al, 1991;Brunt et al, 1992;Modi et al, 1995). This result suggested that our sample did not contain a 1/1 molar ratio of heme/protein.…”

Spectroscopic identification of the haem ligands of cellobiose oxidase

“…Chapman (University of Edinburgh, Scotland) [7], C~,tochrome c (equine heart type VI) was purchased from Sigma, Electronic spectra were recorded on a Hitachi U.2000 spectrephotometer. MCD spectra were recorded on a Jasco J.500 D spectrephotometer, for the wavelensth ranlie 300-1,100 am, and on a bornebuilt circular dichro~mpla in the range 800-3.000 nm.…”

“…These groups are located within protein domains which may be separated by papain cleavage of the linking peptide (el. flavocytochrome b2 [7]) [8,9]. The flavin domain retains its ability to react with cellobiose and is antigenically very similar to ceilobiose quinone oxidoreductase, which can also be isolated from the growth medium of the fungus~ Abbrevmttons: D20/HOD, deuterium oxide/deuterium hydroxide; ~, extinction coefficient; EPR, electron paramagnetic resonance; FAD, florin adenine dinucleotide; His, histidine; MCD, magnetic circular dichroism; M,t, methioniae; NMR, nuclear magnetic r~sonance.…”