Coupled Oxidation of Heme Covalently Attached to Cytochrome b₅₆₂ Yields a Novel Biliprotein

Abstract

A variant of Escherichia coli cytochrome b(562) with covalently attached heme can be converted to a biliverdin-containing protein in two distinct stages by coupled oxidation and acid hydrolysis. The first stage of coupled oxidation yields a stable verdoheme-containing protein. This verdoheme protein is unusual in three respects. First, the verdoheme group is covalently bound to the protein through a c-type thioether linkage. Second, the oxidation stops at the verdoheme stage, and finally, this is the first report of verdoheme generated from a heme protein with exclusive methionine ligation to the heme iron. In addition, the oxidation process does not require denaturation of the protein. The product has been characterized by optical spectroscopy, ESI mass spectrometry, and (1)H NMR. The NMR data show that the predominant product is the result of oxidation at the alpha-meso carbon. A collective evaluation of data on the topic suggests that the electronic structure of the heme, not protein steric effects, is the main factor in controlling the regiospecificity of the oxidation site. In the second stage of conversion to a biliprotein, we demonstrate that the verdoheme ring can be opened by treatment with aqueous formic acid to give alpha-biliverdin covalently attached to the folded protein. This product, a protein-bound linear tetrapyrrole as characterized by optical spectroscopy and mass spectrometry, is an example of a phycobilin chromophore that has not been observed previously.

ThehmuQandhmuDGenes fromBradyrhizobium japonicumEncode Heme-Degrading Enzymes

“…The reaction product had a major atomic peak (m/z) at 583.4, which was identical to the biliverdin standard (data not shown). Nonenzymatic cleavage of heme by coupled oxidation does not result in biliverdin production (17). These data show that biliverdin is produced by heme degradation catalyzed by HmuQ, which is expected for a heme oxygenase.…”

IsdG and IsdI, Heme-degrading Enzymes in the Cytoplasm of Staphylococcus aureus

“…IsdG-or IsdI-mediated heme degradation in the presence of NADPH-cytochrome P450 reductase or ascorbate was not inhibited by catalase at a ratio of 0.5:1.0 (catalase to hemoprotein). Furthermore, coupled oxidation of heme typically leads to the formation of verdoheme, which remains associated with the hemoprotein (35). Upon addition of pyridine to the hemoprotein-verdoheme complex, verdoheme becomes dislodged from the hemoprotein and associates with pyridine, forming a strong pyridine-verdohemochrome spectrum.…”

“…Non-enzymatic oxidation of heme has been reported for certain heme-binding proteins such as myoglobin, cytochrome b 5 , and cytochrome b 562 (33)(34)(35). The coupled binding and oxidation of heme requires exogenous hydrogen peroxide, and therefore can be inhibited by catalase (34,36).…”

A Cytochrome b562 Variant with a c-Type Cytochrome CXXCH Heme-binding Motif as a Probe of the Escherichia coli Cytochrome c Maturation System

“…NMR spectra were collected as described previously (16,30,31) using a Bruker AMX 500 spectrometer or a Bruker Avance 700 spectrometer. The NMR methods used with the different instruments were similar, except that WATERGATE sequences were used on the 700-MHz instrument to remove the residual water signal.…”

“…The NMR methods used with the different instruments were similar, except that WATERGATE sequences were used on the 700-MHz instrument to remove the residual water signal. All spectra were acquired at 300 K. Data processing was as described previously (16,30,31), except that it was performed using XWINNMR (Bruker). All samples were between 0.4 and 1 mM protein in 20 mM potassium phosphate buffer in D 2 O, pH* 6.6, and 0.5 M KCl.…”