Replacement of heme with other metalloporphyrins has been a useful tool for studying functions and properties of proteins ranging from hemoglobin and myoglobin to cytochrome P450cam
and soluble guanylyl cyclase.43, 50, 51, 53
This tool was applied to investigate the role of heme in proper folding and expression of CBS.59
When CBS is expressed in a heme biosynthesis-deficient yeast strain, the protein does not accumulate. CBS expression is rescued by addition of a chemical chaperone, or by protoporphyrin IX and metallated derivatives. Modest yields of active Mn hCBS and Co hCBS proteins could be isolated by expression in anaerobically grown heme deficient E. coli
Recently, we developed an aerobic expression method that enables isolation of large quantities of high purity Co hCBS sufficient for spectroscopic study.29, 30
This Co hCBS is of particular interest because it is virtually indistinguishable from WT Fe hCBS in terms of its native conformation, enzyme activity, AdoMet responsiveness, PLP saturation, and ability to process alternative substrates.29, 30
These prior expression results implicated a structural role for the CBS heme; the availability of large quantities of Co hCBS enables us to identify the similarities and differences between the Co and Fe coordination environments that may contribute to the structural significance of the heme in CBS. To date, there is still no definitive consensus on heme's function in CBS; this puzzle was the impetus for characterization of the various redox and ligation states of the Co-substituted variant of hCBS ().
The spectral features of Co(III) hCBS suggest that the cobalt retains the same coordination environment (Cys/neutral donor, presumably His) as iron in WT Fe(III) hCBS (Cys/His).10
The similarities between Co(III) hCBS and Fe(III) hCBS highlight the structural importance of the metal-cysteine(thiolate) bond in the function of the CBS heme. First, thiolate coordination to the metal center of the porphyrin in CBS is robust and not metal-specific. That is, thiolate coordination persists when cobalt is substituted in lieu of iron, and the enzyme does not distinguish between the two metals. This identical coordination environment manifests itself spectroscopically and allows for high, AdoMet responsive enzymatic activity (both canonical and alternative) even in the presence of Co(III) PPIX (). In contrast, Hg-reacted Fe(III), Hg-reacted Co(III)- and Co(II) hCBS, which do not bear the thiolate ligand, exhibited impaired activities and are non-responsive to AdoMet. From these data it is evident that a metal-thiolate bond is essential for normal CBS function, but the metal does not have to be iron. Furthermore, the slow rate and the odd pH dependence of Co(III) reduction to form Co(II) hCBS suggest a complex process that ultimately results in loss of the cysteine(thiolate) ligand. Previously, heme in CBS was postulated to function as a redox regulator;60
however, the behavior of Fe(II) hCBS is incompatible with a redox regulatory process.23
Fe(II) hCBS is unstable and undergoes slow loss of activity due to replacement of the cysteine(thiolate) ligand by an unidentified neutral donor ligand.23
The behavior of Co hCBS, with its very slow reduction, concomitant loss of the thiolate ligand and minimal activity in the Co(II) state is similarly incompatible with redox regulation.
A second implication is that substitution of Co for Fe likely does not disturb the secondary coordination sphere of the porphyrin in CBS. Earlier work has shown that electrostatic interaction of Arg266
with the axial ligand Cys52
in the WT Fe(III) hCBS is integral to maintaining maximal activity.49
The proposal was made that changes in the heme coordination environment, in particular at Cys52
, may be transmitted through the enzyme to the active site PLP cofactor ~ 20 Å away. The fact that Co(III) hCBS is fully active suggests that this essential secondary coordination sphere is intact. Without this essential electrostatic interaction, the activity of Hg-reacted Co(III) and Co(II) hCBS is substantially altered. Furthermore, the identical coordination environment and unaffected activity of Co(III) hCBS seems incompatible with the possibilities that the Co porphyrin is bound in a different location in the polypeptide chain or that the enzyme itself is folded improperly. These results suggest that there is a specific structure required for activity, which is defined by the presence of the metalloporphyrin and the specific ligands that are bound.
That the N-terminal heme helps to define the structure of CBS is supported by a variety of evidence. An N-terminal deletion variant lacking heme exhibited low enzymatic activity (~20% of WT);7
however, the origin of the low activity was not explored. The activity defect may be due to the absence of heme, misfolding, or both. The absence of heme in lower eukaryotic CBS proteins and mechanistic studies of hCBS, clearly eliminate any role for heme in catalysis.17-19
Furthermore, when hCBS is expressed in heme deficient cells, minimal activity is observed. Modest activity restoration is obtained when Fe-, Mn-, Co-, Sn- and ZnPPIX or metal free PPIX are added to the growth medium. Near complete restoration of activity is observed when the chemical chaperone, trimethylamine N-oxide, is added.59
These observations suggest that proper folding of the enzyme during expression is essential, but that the correctly folded structure may be obtained in a variety of ways. Once properly folded around FePPIX, subsequent disruption of the iron-cysteine(thiolate) ligand bond causes a loss of enzyme activity, which is presumed to result from a change in structure.16, 22, 23, 25, 27
These data, when combined with the fact that hCBS does not discriminate between Fe and Co, together support a structural role of the heme in CBS; perturbation of metal identity in the metalloporphyrin appears not to modify enzyme activity so long as the enzyme is folded correctly and/or the proper metal ligands are maintained.