Table 2.
Determination of factors required for P-cluster maturation
| Line | Assay components |
Activities |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Av1 | NifZ | Av2wt | ATP | FeMoco | C2H4 formation under C2H2/Ar | H2 formation under Ar | NH3 formation under N2 | H2 formation under N2 | Average, % | |
| 1 | Av1ΔnifB* | − | − | − | + | 1,014 ± 29 (100) | 998 ± 31 (100) | 633 ± 6 (100) | 223 ± 34 (100) | 100 |
| 2 | Av1ΔnifZΔnifB† | − | − | − | + | 504 ± 4 (50) | 545 ± 51 (55) | 311 ± 7 (49) | 108 ± 4 (48) | 51 |
| 3 | Av1ΔnifZΔnifB | + | + | + | + | 864 ± 33 (85) | 888 ± 39 (89) | 582 ± 22 (92) | 173 ± 7 (78) | 86 |
| 4 | Av1ΔnifZΔnifB | − | + | + | + | 525 ± 6 (52) | 576 ± 12 (58) | 276 ± 50 (44) | 118 ± 2 (53) | 52 |
| 5 | Av1ΔnifZΔnifB | + | − | + | + | 520 ± 8 (51) | 550 ± 21 (55) | 325 ± 51 (51) | 105 ± 1 (47) | 51 |
| 6 | Av1ΔnifZΔnifB | + | + | − | + | 520 ± 42 (51) | 567 ± 16 (57) | 317 ± 52 (50) | 113 ± 7 (51) | 52 |
| 7 | Av1ΔnifZΔnifB | − | − | + | + | 485 ± 7 (48) | 545 ± 15 (55) | 288 ± 54 (45) | 110 ± 1 (49) | 49 |
| 8 | Av1ΔnifZΔnifB | − | + | − | + | 490 ± 17 (48) | 559 ± 12 (56) | 323 ± 50 (51) | 111 ± 1 (50) | 51 |
| 9 | Av1ΔnifZΔnifB | + | − | − | + | 493 ± 15 (49) | 537 ± 19 (54) | 302 ± 58 (48) | 111 ± 2 (50) | 50 |
Activities of C2H4 formation under C2H2/Ar, H2 formation under Ar, NH3 formation under N2, and H2 formation under N2 are expressed as nanomoles per minute per milligram of protein. Percentages relative to Av1ΔnifB (line 1) are given in parentheses. The assays were performed as described in Materials and Methods. The lower detection limits for the activities were 0.01, 0.02, 0.001, and 0.02 nmol·min−1·mg protein−1 for C2H4 formation under C2H2/Ar, H2 formation under Ar, NH3 formation under N2, and H2 formation under N2, respectively.
*Av1ΔnifB contains 100% fully assembled P-cluster and can be fully activated upon insertion of isolated FeMoco (17).
†Av1ΔnifZnifB contains 50% fully assembled P-cluster and can be activated to 50% of that of Av1ΔnifB in the absence of P-cluster maturation factors (19).