Heme replacement with another metalloporphyrin or a porphyrin with a different peripheral side chains represents a powerful approach for elucidating heme function in proteins. Typically, reconstitution of apoenzymes with heme analogs is an effective method for preparation of substituted hemoproteins (reviewed in [32
]); however, this approach has been limited to proteins that can withstand heme removal and its replacement under partially denaturing conditions. Therefore, a different approach should be utilized for heme replacement in heme proteins sensitive to partial denaturation and/or simultaneous removal of other cofactors. Cystathionine β–synthase (CBS) of higher organisms is the only PLP-dependent enzyme known so far that also contains a heme (FePPIX) moiety. CBS condenses serine and homocysteine to form cystathionine, via a classic β–replacement reaction attributed to the PLP cofactor [3
]. The heme in CBS is not essential for the catalytic reaction, nor is the heme involved in redox regulation or ligand binding [7
]. Thus, the important question that remains unresolved is why CBS enzymes of higher organisms contain a heme (FePPIX) moiety.
Our previous attempt at heme replacement in CBS employed an E. coli
strain deficient in heme biosynthesis [20
]. Growing this strain anaerobically eliminated the need for heme. The medium was supplemented with heme analogs such as MnPPIX or CoPPIX. CBS expressed and purified under these conditions had significantly lower specific activity. The yield of the enzyme was also low thus preventing detailed characterization. In this communication, we report expression, purification and characterization of an aerobically expressed CoCBS in iron-depleted cells in the presence of CoCl2
. This procedure yielded large amounts of CoCBS indistinguishable in activity from wild type FeCBS.
Recently, we showed that repeated growing of E. coli
cells in a minimal medium supplemented with cobalt chloride resulted in the introduction of cobalt into protoporphyrin IX and subsequently in the incorporation of CoPPIX into heme proteins [21
]. This approach allowed for more convenient cell cultivation and protein expression using a standard or an engineered E. coli
expression strain, such as Rosetta 2 (DE3) employed in the present study [20
]. The presence of pRARE2 plasmid in E. coli
Rosetta 2 (DE3), which supplies tRNAs for 7 rare codons and thus enhances the expression of eukaryotic proteins, appeared to have a beneficial influence on two aspects of CBS expression and purification: (i) increased abundance of full-length polypeptide compared to the most likely prematurely terminated polypeptides and (ii) concomitantly decreased presence of CBS antibody positive material in the insoluble fraction.
The CoCBS was expressed as a fusion protein with a GST partner at the N-terminus, which was subsequently cleaved off with PreScission protease leaving a single extra amino acid residue, glycine, at the N-terminus of CBS [22
]. The DEAE Sepharose step was subsequently used for the separation of GST and CoCBS. The aggregative tendencies of the human full length CBS often led to protein precipitation during concentration and thus resulting in a great variability in yields [5
]. The presence of non-ionic detergent (0.01% Tween 20) in the protein exchange buffer decreased the aggregation of wild type FeCBS as well as CoCBS during concentration.
CoCBS, with CoPPIX replacing heme, was isolated on expression in cobalt-supplemented minimal medium. The results of spectral analysis of CoCBS protein and pyridine hemochromogen assay correlate very well with our previous report on CoCBS prepared by an approach employing heme-deficient E. coli
]. The metal analysis by using ICP-OES confirmed high-level substitution of iron by cobalt in CoCBS, wherein iron content was minimized to 13.1% in CoCBS 7× and further decreased to only 7.6% in CoCBS 12×. The analytical determination of cobalt content in these CoCBS enzymes correlates with spectrophotometric estimates of CoPPIX and FePPIX content reported previously [21
]. The efficiency of present metalloporphyrin replacement method correlates well with previously employed total porphyrin replacement method utilizing a heme-deficient E. coli
strain: the iNOSheme
expressed in the presence of MnPPIX contained ≤ 5% of iron contamination [35
The most striking difference between CoCBS described here and the one prepared by employing CBS expression in heme biosynthesis-deficient E. coli
in the presence of CoPPIX [20
] is the enzymatic activity data. We found CoCBS practically indistinguishable from wild type FeCBS for two different enzymatic activities. Importantly, the protein used in these assays is pure and contains >90% Co; clearly, the high activity is not due to residual FeCBS. The low activity of CoCBS reported previously must have been a consequence of the method employed to obtain the protein. The high activity of CoCBS reported herein demonstrates not only that heme (FePPIX) is not essential for the catalytic activity of CBS, but also that it can be replaced by other metalloporphyrin (such as CoPPIX) without any decrease of enzymatic activity. The fact that a non-native metal enables full activity supports the notion that porphyrin moiety in CBS performs a structural role.