Structure: Function Studies of the Cytosolic, Mo- and NAD+-Dependent Formate Dehydrogenase from Cupriavidus necator

Abstract

<jats:p>Here, we report recent progress our laboratories have made in understanding the maturation and reaction mechanism of the cytosolic and NAD+-dependent formate dehydrogenase from Cupriavidus necator. Our recent work has established that the enzyme is fully capable of catalyzing the reverse of the physiological reaction, namely, the reduction of CO2 to formate using NADH as a source of reducing equivalents. The steady-state kinetic parameters in the forward and reverse directions are consistent with the expected Haldane relationship. The addition of an NADH-regenerating system consisting of glucose and glucose dehydrogenase increases the yield of formate approximately 10-fold. This work points to possible ways of optimizing the reverse of the enzyme’s physiological reaction with commercial potential as an effective means of CO2 remediation. New insight into the maturation of the enzyme comes from the recently reported structure of the FdhD sulfurase. In E. coli, FdhD transfers a catalytically essential sulfur to the maturing molybdenum cofactor prior to insertion into the apoenzyme of formate dehydrogenase FdhF, which has high sequence similarity to the molybdenum-containing domain of the C. necator FdsA. The FdhD structure suggests that the molybdenum cofactor may first be transferred from the sulfurase to the C-terminal cap domain of apo formate dehydrogenase, rather than being transferred directly to the body of the apoenzyme. Closing of the cap domain over the body of the enzymes delivers the Mo-cofactor into the active site, completing the maturation of formate dehydrogenase. The structural and kinetic characterization of the NADH reduction of the FdsBG subcomplex of the enzyme provides further insights in reversing of the formate dehydrogenase reaction. Most notably, we observe the transient formation of a neutral semiquinone FMNH·, a species that has not been observed previously with holoenzyme. After initial reduction of the FMN of FdsB by NADH to the hydroquinone (with a kred of 680 s−1 and Kd of 190 µM), one electron is rapidly transferred to the Fe2S2 cluster of FdsG, leaving FMNH·. The Fe4S4 cluster of FdsB does not become reduced in the process. These results provide insight into the function not only of the C. necator formate dehydrogenase but also of other members of the NADH dehydrogenase superfamily of enzymes to which it belongs.</jats:p>

The Reversible Electrochemical Interconversion of Formate and CO2 by Formate Dehydrogenase from Cupriavidus necator

“…The smallest subunit FdsD bears no redox-active cofactors but is implicated in Mo cofactor insertion . The X-ray crystal structure of the FdsBG subcomplex from C. necator has previously been determined, demonstrating that the FMN cofactor (of FdsB) is the site at which reduction of NAD + occurs, as is known to be the case in other members of the NADH dehydrogenase superfamily of enzymes, of which FdsDABG is a member. ,− An unusual and convenient property of FdsDABG is its relatively high stability under aerobic conditions, although stabilizing inhibitors such as potassium nitrate or sodium azide are required for long-term retention of activity …”

Redox Characterization of the Complex Molybdenum Enzyme Formate Dehydrogenase from Cupriavidus necator

“… a The numbering of the Fe/S clusters (A1, A2, ... B6, G7) is taken from the published cryo-EM structure of FdsDABG ( R. capsulatus ) while the EPR signal numbering (Fe/S 1 – Fe/S 5 ) is taken from previous EPR studies. , . …”

“…An EPR signal for the His-coordinated [4Fe−4S] cluster (A4, Figure 1) remains unidentified. X-band hyperfine sublevel correlation (HYSCORE) spectra were analyzed to see if the expected strong 14 N hyperfine couplings (A( 14 N) ∼ 7−10 MHz) that typically arise in His coordinated Fe/S clusters are present in any of the EPR components. 46−48 Figure 13 shows a HYSCORE spectrum measured at 10 K and 345 mT, the 19 We conclude that the EPR spectrum attributable to Fe/S A4 has yet to be observed.…”

“…The numbering of the Fe/S clusters (A1, A2, ... B6, G7) is taken from the published cryo-EM structure of FdsDABG (R. capsulatus) 15 while the EPR signal numbering (Fe/S 1 − Fe/S 5 ) is taken from previous EPR studies. 14,17 . The complexity of the optical spectroelectrochemistry of FdsDABG, with FMN and seven Fe/S clusters as chromophores (Figures S3 and S4), presents significant challenges in data analysis.…”

“…However, this is not a universal trait. The bacterial Mo-dependent formate dehydrogenase FdsDABG from Cupriavidus necator exhibits excellent air stability and has already been explored in biotechnological applications. − The cryo-EM structure of FdsDABG from Rhodobacter capsulatus , which is highly homologous with FdsDABG from C. necator , has provided a clear picture of the relative orientations of the redox cofactors. The enzyme is a heterotetramer with the FdsA (105 kDa), FdsB (55 kDa), and FdsG (19 kDaA) subunits all bearing redox-active cofactors, while the small FdsD subunit (7 kDa), which has no cofactors, is thought to be involved in Mo factor insertion …”